Isolation and identification of heavy metal-tolerant bacteria from an industrial site as a possible source for bioremediation of cadmium, lead, and nickel.
Heavy metal pollution is identified as one of the consequences brought about by development and economic progress. The continuous discharge of substances, particularly heavy metals brought about by these developments, has created a toll on both the environment and the organisms exposed. The extensive discharge and persistence of heavy metals brings about the concern, especially that it affects both the environmental quality and the existence and condition of the organisms. The problem of heavy metal pollution is evident not only in the country but in other parts of the world. The persistence of heavy metals brings a global concern especially that possible consequences occur even in exposures to small concentrations.
Several methods have been explored to mitigate the effects of heavy metals and to control the occurrence and persistence of these substances in the environment. Most of the methods being employed tend to be expensive, and developing countries encounter difficulties in its implementation because of the exorbitant costs associated to such treatments and/or measures. Currently, bioremediation is being explored as an effective and technological solution to the problem brought about by heavy metal pollution. Studies have shown that bioremediation using plants [1,2] and microorganisms  have that potential to remove, degrade, and/or inactivate heavy metals. The problem with the use of phytoremediation is that it is time-consuming and is dependent on a variety of factors that include soil chemistry, root depth, amount of biomass produced, type of plant, level of contamination and its concentrations, climate, and the impacts of the contaminants to the plant . There is a need to consider measures that can help clean up and remove the harmful heavy metals persisting in the environment with little disruption of its surrounding environment. There is a need to screen and find indigenous microorganisms that have the potential to help solve the problem brought about by heavy metal pollution. To the best of our knowledge, there are few bacterial strains reported to degrade heavy metals in the Philippines. There is a paucity of information on particular microorganisms that can resist and are tolerant to heavy metals. This study aimed to isolate bacterial microorganisms from an industrial site and screen for their potential to bioremediate heavy metals such as cadmium, lead, and nickel and identify the bacterium morphologically, biochemically, and molecularly . Results of this study are vital, as they provide baseline information on the microorganisms that have the capacity to resist and tolerate heavy metals and the potential for bioremediating heavy metal-contaminated environments.
Study area and sample collection:
Surface soil samples were obtained in three industrial sites situated in Marikina City, Philippines. Soil samples were collected from the surface up to a depth of 15 cm, after which they were placed in sterile Ziplock bags using a sterile spatula. All samples were stored at 4[degrees]C and transported to the laboratory for immediate processing .
Chemicals and media:
Stock solutions of the heavy metals (1000 mg/L) were prepared. The metal salts used were Cd[Cl.sub.2], Pb[(N[O.sub.3]).sub.2], and NiS[O.sub.4] (Merck, Germany). Nutrient agar and Luria Bertani (LB) medium were used to grow and isolate the microorganisms.
Isolation of bacteria:
One gram of soil sample obtained from each site was suspended in 10 ml of sterile distilled water and vortexed for 1 min. Soil suspensions were centrifuged at 2000 rpm for 10 min, and 0.1 ml of the supernatant was diluted up to [10.sup.-5] dilutions, plated on nutrient agar in triplicates, and incubated at 37[degrees]C for 24 h. Individual colonies of bacteria that showed varied shapes and color were selected, picked off, and purified on subsequent transfers to nutrient agar . The pure bacterial isolates were maintained on nutrient agar at 4[degrees]C and re-cultured every 4 weeks.
Screening of heavy metal resistance:
The isolated bacterial colonies were screened for heavy metal resistance to cadmium, lead, and nickel by adding 50 [micro]g/ml of Cd[Cl.sub.2], Pb[(N[O.sub.3]).sub.2], and NiS[O.sub.4], respectively, to sterilized nutrient agar medium. The isolated bacterial colonies were spot inoculated onto the nutrient agar plates  and incubated at 37[degrees]C for 24 h.
Maximum tolerable concentration of bacterial isolates:
The maximum tolerable concentration is the highest concentration of the heavy metal that allows growth after 2 days . Isolated bacterial colonies that grew initially on the nutrient agar supplemented with the heavy metals cadmium, lead, and nickel were exposed to increasing heavy metals concentrations (50, 150, 300, 450, 600, 750, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100 pg/ml). Testing for tolerance of the microorganisms ended when complete inhibition of the growth was observed on the nutrient agar with metal supplementation .
Growth study of metal resistant isolates:
Metal-resistant isolates obtained in nutrient agar supplemented with metal were grown in LB broth supplemented with 100 [micro]g/ml of the heavy metals cadmium, lead, and nickel incubated at 37[degrees]C for 24 h and agitated on a rotary shaker (150 rev/min) , . Growth was monitored as a function of biomass by measuring absorbance at 600 nm using spectrophotometer (Dynamica Halo RB-110). Growth of the isolates on LB with no metal supplementation (control) and with metal supplementation (test) were performed and compared by plotting the optical density at 600 nm ([OD.sub.600nm]) to time in hours.
Bacterial colonies isolated from the nutrient agar with metal supplementations were morphologically characterized through their cultural characteristics, Gram staining, endospore staining, and biochemical characterization.
Isolation of bacterial isolates using 16s rRNA:
Bacterial colonies isolated from the nutrient agar with metal supplementations were characterized molecularly using the 16s rRNA sequencing. The genomic DNA was extracted using a Vivantis GF-1 Bacterial DNA extraction kit (Vivantis Technologies Sdn. Bhd., Malaysia) according to the manufacturer's instructions . The extracted DNA was then used as a template for PCR to amplify the 16s rRNA gene. The forward primer EUB f933 (5'-GC-clamp-GCACAAGCGGTGGAGCATGTGG-3') and reverse primer EUB r1387 (5'-GCCCGGGAACGTATTCAcCG-3') were used to amplify the 16s rRNA gene . PCR was performed with a 50-[micro]l reaction mixture containing the DNA extract as a template, each primer at a concentration of 5 mM, 25 mM Mg[Cl.sub.2], and dNTPs at a concentration of 2 mM and 1.5 U of Taq polymerase and buffer. The PCR conditions were as follows: initial denaturation for 5 min at 94[degrees]C, followed by 30 cycles of denaturation at 94[degrees]C for 30 s, annealing at 55[degrees]C for 30 s, extension at 72[degrees]C for 90 s, and final extension at 72[degrees]C for 10 min. PCR was carried out using the Quanta Biotech (SI-96). The PCR products were analyzed by 1.5% (w/v) agarose gel electrophoresis in 1 x TAE buffer with ethidium bromide (0.5 pg/ml) using the Mupid-One. Samples for sequencing were sent to MACROGEN, Korea. Sequences were compared with the available sequences against the 16s rRNA sequences database using NCBI's BlastN. Sequences were aligned using ClustalW program in Mega 6.0. Similarity index was generated and compared with known sequences.
RESULTS AND DISCUSSION
A total of 600 bacterial colonies were isolated from the soil samples obtained from the three industrial sites. Of the total bacterial colonies isolated and grown in nutrient agar with metal supplements, two bacterial colonies (B1, B2) were resistant to the heavy metal cadmium, one bacterial colony (B3) was resistant to the heavy metal lead, and three bacterial colonies (B4, B5, B6) were resistant to the heavy metal nickel. No microorganism showed resistance to two or more metals. All the bacterial colonies that were lead-resistant were tolerant up to 2000 [micro]g/ml of Pb(N[O.sub.3]) (2), whereas the bacterial colonies resistant to cadmium and nickel were tolerant up to 1200 [micro]g/ml of Cd[Cl.sub.2] and NiS[O.sub.4], respectively. Different microorganisms showed varied responses to the metals as observed from the study . The six isolated colonies capable of growing in the heavy metals cadmium, lead, and nickel have that potential in bioremediation and as bioindicator of heavy metal pollution. The microorganisms also showed varied tolerances to the metals. A study  indicated that heavy metals have similar toxic mechanisms, but the possibility of tolerances for metal resistant microorganisms varies. It is likely that it varies because microorganisms have mechanisms of resistance to different metals , which may include complexation, binding with cell envelopes, metal reduction, and metal efflux .
Comparative analyses of the sequences from the NCBI databases showed that the strains were closed to the members of the genera Bacillus, Chryseobacterium, and Pseudomonas. The highest sequence similarities of the group are B1, Bacillus cereus (98% similarity to Bacillus cereus strain BDBC01, accession number JX276537.1); B2, Bacillus amyloliquefaciens (98% similarity to Bacillus amyloliquefaciens strain D4F1, accession number KC894960.1); B3, Bacillus subtilis (92% similarity to Bacillus subtilis strain KYLS-CU03, accession number KF111802.1); B4, Chryseobacterium sp. (99% similarity to Chryseobacterium sp. strain TH1, accession number JN208181.1); B5, Pseudomonas aeruginosa (99% similarity to Pseudomonas aeruginosa strain KF539786.1, accession number KF539786.1); and B6, Bacillus aerius (99% similarity to Bacillus aerius strain RGS230, accession number KC469617.1).
Table 1 shows the morphological and biochemical characteristics and molecular identification of the bacterial colonies isolated on the nutrient agar with metal supplementation including their identification through molecular characterization using the 16s rRNA. Four organisms were Gram-positive rods, and two were Gramnegative rods.
This experiment has shown that the Bacillus cereus and Bacillus amyloliquefaciens were particularly resistant to Pb, Bacillus subtilis was resistant to Cd, and Chryseobacterium sp., Pseudomonas aeruginosa, and Bacillus aerius were resistant to Ni. The growth of the microorganisms showed varied results in LB with metal supplementations compared to those grown in LB without metal supplementations as shown in Figure 1. Our results are corroborated by other studies where metals may have an influence on the growth of microorganisms [11,12]. Higher growth was evident in the microorganisms in LB with Ni. This result is likely as Ni plays an important role in the metabolic process of the microorganism . A study  presents that the reduction in the growth of stressed microorganisms to metals may be likely that microorganisms have to deviate their energy from growing to maintaining their cellular functions so as to resist metal toxicity.
In this study, we found six bacterial isolates that were resistant to the heavy metals cadmium, lead, and nickel. No microorganism showed multiresistance to two or more metals. The metal-resistant microorganisms isolated and identified likewise showed reduced growth in media supplemented with metals compared to the control and had varied metal tolerances. The Bacillus cereus and Bacillus amyloliquefaciens had the highest metal tolerance compared to the other microorganisms isolated. Results of the study suggest that the metal-resistant microorganisms isolated can be potentially useful for the remediation of heavy metals-contaminated environments.
Received 28 December 2015; Accepted 28 January 2016; Available online 24 February 2016
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(1) Margaret L.C. De Guzman, (2) Kristina Samantha G. Arcega, (2) Jan-Mari Norbertine R. Cabigao, (1) Glenn L. Sia Su
(1) Biology Department, University of the Philippines-Manila, Manila, Philippines
(2) Biology Department, Miriam College, Quezon City, Philippines
Address For Correspondence:
Margaret L.C. De Guzman, Department of Biology, College of Arts and Sciences, University of the Philippines-Manila, Padre Faura Manila, Philippines.
Tel: +63 2 5265861; E-mail: email@example.com
Table 1: Biochemical and morphological characterization and molecular identification of the metal-resistant bacterial isolates in an industrial site. Bacteria B1 B2 B3 Molecular Bacillus Bacillus Bacillus Identification cereus amyloliquefaciens subtilis Morphological (+) (+) (+) Gram stain White White Cream Color Rods Rods Rods Cell morphology (+) (+) (+) Motility (+) (+) (+) Endospore Irregular Irregular Irregular Margin Biochemical (+) (+) (+) Catalase (-) (+) (-) Oxidase (+) (-) (+) Citrate Bacteria B4 B5 B6 Molecular Chryseobacterium Pseudomonas Bacillus Identification sp. aeruginosa aerius Morphological (-) (-) (+) Gram stain Yellow Bluish green White Color Rods Rods Rods Cell morphology (+) (+) (+) Motility (-) (-) (+) Endospore Irregular Irregular Irregular Margin Biochemical (+) (+) (+) Catalase (+) (+) (+) Oxidase (-) (+) (+) Citrate
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|Author:||De Guzman, Margaret L.C.; Arcega, Kristina Samantha G.; Cabigao, Jan-Mari Norbertine R.; Su, Glenn L|
|Publication:||Advances in Environmental Biology|
|Date:||Jan 1, 2016|
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