Heavy metal and antibiotic resistance of ureolytic bacteria and their immobilization of heavy metals

Ecological Engineering 97 (2016) 304–312

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Heavy metal and antibiotic resistance of ureolytic bacteria and their immobilization of heavy metals Chang-Ho Kang, Jae-Seong So ∗ Department of Biological Engineering, Inha University, Incheon 22212, Korea

a r t i c l e

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Article history: Received 11 February 2016 Received in revised form 19 July 2016 Accepted 5 October 2016 Keywords: Heavy metal Antibiotic Resistance Microbially induced calcite precipitation (MICP) Immobilization

a b s t r a c t Environmental pollution by toxic heavy metals is spreading worldwide along with industrial progress. The isolation and characterization of microorganisms capable of resisting elevated concentrations of heavy metals as well as multiple types of antibiotics are critical to the development of an effective bioremediation strategy for polluted sites. In this study, we first investigated the interplay between heavy metals and the antibiotic resistance of ureolytic bacteria. The antibiotic resistance patterns revealed that the heavy metal resistance of these isolates was closely associated with their resistance to antibiotics. In addition, we examined the immobilization of heavy metals by these isolates, based on microbially induced calcite precipitation (MICP). The unconfined compressive strength of a cylinder specimen injected once with the selected bacterial culture showed a 3.7-fold increase relative to an untreated specimen. In addition, it was found that the heavy metals were highly immobilized in the bacteria-treated cylinder samples. © 2016 Published by Elsevier B.V.

1. Introduction A worldwide environmental problem has developed over the past few decades owing to the rapid increase in industrialization and urbanization. Elevated concentrations of heavy metals are introduced into the environment through metalliferous mining, metal smelting, activities of metallurgical industries, waste disposal, and corrosion of metals in use (Bachate et al., 2013; Fan et al., 2014; Prithviraj et al., 2014; Kang et al., 2015). Several methods for treating environmental contaminants have been developed over the past decades, such as applying physical, chemical, and biological processes. Although physical and chemical approaches are capable of removing a broad spectrum of contaminants, the main disadvantages of these methods lie in their increased energy consumption and need for additional chemicals (Máthé et al., 2012). The use of microorganisms to sequester, precipitate, or alter the oxidation state of various heavy metals (Rittle et al., 1995) through reduction, accumulation, mobilization, and immobilization (Lovley, 1994; Avery, 1995; Valentine et al., 1996; Kang et al., 2014) has been studied in several countries. During bioremediation, the metabolic activity of microorganisms is involved in the breakdown of contaminants into non-toxic compounds. This tech-

∗ Corresponding author at: Department of Biological Engineering, Inha University, 100 Inha-ro, Nam-gu, Incheon 22212, South Korea. E-mail address: [email protected] (J.-S. So). http://dx.doi.org/10.1016/j.ecoleng.2016.10.016 0925-8574/© 2016 Published by Elsevier B.V.

nique is cost-effective, is applicable over large areas, and can lead to the complete breakdown of the organic contaminants, potentially ending in their mineralization (Sarkar et al., 2004; Trindade et al., 2004). Microbially induced calcite precipitation (MICP)-based degradation of urea occurs through the ureolytic pathway, which produces ammonium ions as an energy source and leads to the alkalization of the surrounding environment (Whiffin et al., 2007). In addition to NH4 + , carbonate ions are formed, which precipitate as calcite (CaCO3 ) in the presence of Ca2+ (Hammes and Verstraete, 2002). Moreover, when these reactions occur in sand, crystals are formed between the sand particles and they hold the sand particles together. Carbonate precipitation is an important aspect of biomineralization and has been investigated extensively owing to its wide range of technological applications (Ivanov and Chu, 2008; Anbu et al., 2016). The hydroxide ions result in an increase in pH, which in turn can shift the bicarbonate equilibrium, resulting in the formation of carbonate ions. This shift can then precipitate heavy metal ions in wastewater or soil (Hoffman and Deccho, 1999). The introduction of certain concentrations of heavy metals into the environment kills the majority of the microflora, thereby selecting for a few cells that have evolved resistance mechanisms to the heavy metals. The resistance mechanisms used by microorganisms to tolerate heavy metal stress include permeability barriers, intra- and extracellular sequestration, efflux pumps, enzymatic detoxification, and reduction (Nies, 1999). In some cases, resistance to metal ions has been reported to be plasmid-mediated and

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observed to be encoded by genes in close proximity to antibiotic resistance genes (Alonso et al., 2000; Spain, 2003). A correlation between heavy metal tolerance/resistance and antibiotic resistance in Escherichia coli (Spain, 2003) and Staphylococcus sp. (Groves and Young, 2016) has been reported. Alonso et al. (2000) implicated a cluster of genes to be involved in the antibiotic and heavy metal resistance of a clinical isolate of the gram-negative bacterium Stenotrophomonas (Xanthomonas) maltophilia. Therefore, heavy metal and antibiotic resistance may sometimes be transferred together in the environment. Furthermore, as several bioremediative pathways are also located on mobile genetic elements (e.g., plasmids), a long-term exposure to heavy metals and/or antibiotics may be linked to the widespread distribution of bioremediative capabilities (Roy et al., 2002). In this study, we determined the heavy metal resistance patterns of isolated ureolytic bacteria that showed resistance to elevated concentrations of multiple types of heavy metals. Additionally, the isolated strains were screened for resistance to multiple types of antibiotics. The organisms with a combination of heavy metal and antibiotic resistance would be useful for bioremediation of environments polluted with heavy metals and would also help to overcome metabolic bottlenecks still existing in the bioremediation processes by applying them in processes such as the immobilization of heavy metals.

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(KJ-64), and KC211294 (WJ-2). The cultures were routinely grown at 30 ◦ C and 200 rpm in YA broth (20 g/L yeast extract and 10 g/L ammonium sulfate, pH 7). 2.2. Biochemical and physiological characterization

2. Materials and methods

Biochemical characterization was carried out to detect the presence of enzymes, such as gelatinase, oxidase, galactosidase, and urease, and the utilization of glucose, sucrose, arabinose, among others. Other tests included the indole, Voges–Proskauer, citrate utilization, and H2 S production tests. To select heavy-metal-resistant bacteria, it is necessary to standardize the cultural and physiological conditions of the isolated strains. Among the physicochemical conditions to consider, acid (pH), temperature, and starvation are of great importance to bacterial growth. The isolated strains were grown at different temperatures (5, 20, 30, 40, and 50 ◦ C) in YA broth, with incubation carried out at 200 rpm for 24 h. The strains were also grown in YA broth of different pH values (from 4.0 to 10.0), where incubation was carried out at 30 ◦ C and 200 rpm for 24 h. The growth was measured in terms of optimal density (OD) at 600 nm using a UV/visible spectrophotometer (Ultrospec 2000; Amersham Pharmacia Biotech Inc., NJ, USA). For the starvation test, bacterial cells (1 × 108 CFU/mL) suspended in YA broth were starved at 30 ◦ C for 35 days. The cell viability after the 35 days was determined by the spread plate technique, using triplicate samples and YA agar plates.

2.1. Microbial strains and culture conditions

2.3. Measurement of calcite production and urease activity

Six bacterial strains, isolated from soil from an abandoned mine, were used in this study because of their abilities to induce urease activity, produce calcite, and resist heavy metals (Kang et al., 2014, 2015). These strains were Viridibacillus arenosi B-21 (B-21), Sporosarcina soli B-22 (B-22), Enterobacter cloacae KJ-46 (KJ-46), Enterobacter cloacae KJ-47 (KJ-47), Lysinibacillus sphaericus KJ-64 (KJ-64), and Sporosarcina pasteurii WJ-2 (WJ-2). The 16S rRNA gene sequences determined in this study were deposited in the NCBI GenBank database under the accession numbers KJ671467 (B-21), KJ485701 (B-22), KF598853 (KJ-46), KF598854 (KJ-47), KF598850

One milliliter of a culture of isolated bacteria was grown overnight in BPU broth at 30 ◦ C and 200 rpm for 48 h. The bacterial suspension (500 ␮L) was added to 500 ␮L of calcium chloride dihydrate solution (350 mM). The mixture was centrifuged at 16,179 × g for 5 min at 25 ◦ C to collect the precipitate, which was then dried for 24 h at 50 ◦ C and weighed. Urease activity was determined using the phenol-hypochlorite assay (Natarajan, 1995). The bacterial suspension (250 ␮L) was added to 250 ␮L of sodium phosphate buffer (0.1 M) containing 500 ␮L of urea solution (3 M). The mixture was incubated at 37 ◦ C

Table 1 Biochemical characteristics of isolated strains. Biochemical Tests

Isolated strains B-21

B-22

KJ-46

KJ-47

KJ-64

W-2

Gram reaction 2-nitrophenyl-␤D-galactopyranoside (ONPG) l-arginine (ADH) l-lysine (LDC) l-ornithine (ODC) Trisodium citrate (CIT) Sodium thiosulfate (H2 S) Urea (URE) l-tryptophane (TDA) l-tryptophane (IND) Sodium pyruvate (VP) Gelatin (GEL) d-glucose (GLU) d-mannitol (MAN) Inositol (INO) d-sorbitol (SOR) l-rhamnose (RHA) d-sucrose (SAC) d-melibiose (MEL) Amygdalin (AMY) l-arabinise (ARA)

+ +

+ −

− +

− +

+ +

+ +

− − − − − + − − + − − − − − − − − − −

− − − − − + − − − − − − − − − − − − –

+ − + + − + − − + − + + − + + + + + +

+ − + + − + − − + − + + − + + + + + +

− + − + − + − − + + − − − − − − − − −

− − − − − + − − − + − − − − − − − − −

Identification GenBank Accession number

Viridibacillus arenosi Sporosarcina soli Enterobacter cloacae Enterobacter cloacae Lysinibacillus sphaericus Sporosarcina pasteurii KJ671467 KJ485701 KF598853 KF598854 KF598850 KC211294

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for regular time intervals. Subsequently, 2 mL of phenol nitroprusside solution was added to the alkaline hypochlorite solution and the mixture was incubated at 50 ◦ C for 10 min, following which the absorbance was measured at 626 nm. Ammonium chloride (0–10 ␮M) was used as the standard.

30 s at 94 ◦ C, annealing for 30 s at 50 ◦ C, and extension for 1 min at 72 ◦ C; and a final extension for 7 min at 72 ◦ C. The PCR products were analyzed by 1.5% agarose gel electrophoresis.

2.5. Analysis of heavy metal resistance pattern by maximum tolerance concentration

2.4. PCR amplification of the ureC gene The 323 bp ureC gene amplicons for the six bacterial isolates used in this study were obtained by polymerase chain reaction (PCR). For the amplification, two universal primers were used (Gresham et al., 2007): ureC-F (5 -AAG STS CAC GAG GAC TGG GG3 ) and ureC-R (5 -AGG TGG TGG CAS ACC ATS AGC AT-3 ). PCR amplification was performed under the following conditions: initial denaturation for 5 min at 94 ◦ C; 30 cycles of denaturation for

To determine the maximum tolerance concentration (MTC) of heavy metals of the isolated strains, each isolate was grown at 30 ◦ C and 200 rpm for 24 h in 10 mL of YA broth in screw-capped tubes supplemented with different concentrations of each heavy metal (CoCl2 , CoSO4 ·7H2 O, CuSO4 ·5H2 O, CuCl2 , FeCl3 , FeSO4 , CdCl2 , BaCl2 , PbCl2 , SrCl2 , and ZnSO4 ). Stock solutions of the heavy metals, prepared in distilled water, were sterilized by filtration (0.22 ␮m). From each stock solution, an appropriate amount was diluted in

Fig. 1. Effects of physiological conditions on growth of the isolated strains. (A) pH, (B) temperature, and (C) starvation.

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YA broth to obtain heavy metals in the concentration range of 300–3000 mg/L. Following incubation at 30 ◦ C and 200 rpm for 24 h, each bacterial culture was spread over YA agar plates in duplicates and incubated at 30 ◦ C for 24 h to test for any appearance of growth. The presence or absence of growth was determined visually as being positive or negative, respectively. The absence of bacterial growth indicated its sensitivity to that particular concentration of heavy metal, whereas the presence of growth at a certain heavy metal concentration indicated that the bacterial isolate was resistant to that concentration. 2.6. Analysis of antibiotic resistance pattern Antibiotic sensitivity and resistance of the isolated strains were assayed using the disk diffusion technique (CLSI, 2006). Bacterial suspensions were spread onto Mueller–Hinton agar (Difco, Detroit, MI, USA) plates, onto which antibiotic disks (Oxoid, Basingstoke, Hampshire, UK) were then placed. The plates were incubated at 30 ◦ C for 18–24 h under aerobic conditions. The diameter of the zone of inhibition around each disk was measured and recorded. Each bacterial species was classified as resistant (R), intermediately resistant (I), or susceptible (S), according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2006). The following antibiotics, with their concentrations given in parentheses, were tested: ampicillin (AM; 10 ␮g), cefotaxime (CTX; 30 ␮g), cefotetan (CTT; 30 ␮g), cephalothin (CF; 30 ␮g), chloramphenicol (C; 30 ␮g), ciprofloxacin (CIP; 5 ␮g), cefepime (CEP; 30 ␮g), erythromycin (E; 15 ␮g), gentamicin (GM; 10 ␮g), kanamycin (K; 30 ␮g), nalidixic acid (NA; 30 ␮g), rifampicin (RA; 5 ␮g), tetracycline (TE; 30 ␮g), trimethoprim/sulfamethoxazole (SXT; 1.25 ␮g and 23.75 ␮g, respectively), and vancomycin (VA; 30 ␮g). 2.7. Cylinder experiments of sand The surface percolation treatment method was used in this study (Chu et al., 2012). A cylinder of size ␾ 90 × 15 mm was constructed and treated with a solution of urea, calcium chloride, and heavy metals. Aliquots (25 mL) of the heavy metals (1000 mg/L mixture of CuSO4 ·6H2 O and ZnSO4 ·7H2 O) and 80 g of sand was mixed

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with 25 mL of 5 mM urea and 25 mM calcium chloride, respectively. After 24 h of bacterial cultivation, 25 mL of bacterial suspension (1 × 108 CFU/mL) was injected from the top of the cylinder samples to the bottom, and the cylinder samples were then incubated at 30◦ for 28 days. In the control experiment, no bacterial cells were added. The suspension/solution was then drained off by gravity. After treatment with the bacterial strains, 25 mL of 5 mM urea and 25 mM calcium chloride was overlaid on the cylinder samples for a period of 7 days. When the treatment was finished, the cylinder samples were subjected to tests for determining the cell survivability, compressive strength, and concentration of heavy metals. The compressive strength test determined the compressive strength of the cylinder samples against vertical loading and was performed using a hardness meter (DIK-5553, Daiki, Japan). The specimen was placed into the machine and gradually loaded until it failed (cracked). The ultimate load was recorded, where values are expressed in MPa. The concentrations of heavy metals in the cylinder samples were determined by using an inductively coupled plasma optical spectrometer (Perkin-Elmer OPTIMA-7300DV; PerkinElmer, Inc., Waltham, MA, USA). X-Ray diffraction (XRD) analysis was used to construct a crystal structure model for calcium carbonate. The samples were then analyzed using a DMAX-2500 measurement system (Rigaku, Tokyo, Japan).

3. Results 3.1. Biochemical and physiological characterization The biochemical characteristics of the isolated strains are shown in Table 1. The KJ-46 and KJ-47 strains showed positive results for the utilization of l-arginine, l-ornithine, d-glucose, d-mannitol, d-sorbitol, l-rhamnose, d-sucrose, d-melibiose, amygdalin, and larabinose. The bacterial response to the applied environmental conditions (pH, temperature, and starvation) is important for bioremediation because it is directly related to the survival and growth of bacteria in contaminated sites. The ground waters in Korea are on aver-

Fig. 2. Urease activity and calcite production of Viridibacillus arenosi B-21, Sporosarcina soli B-22, Enterobacter cloacae KJ-46, E. cloacae KJ-47, Lysinibacillus sphaericus KJ-64, and S. pasteurii WJ-2 (values are the mean ± SD).

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age slightly acidic or of neutral pH 6–8 and have poor nutritional conditions. Therefore, the experiments were carried out in consideration of the pH, temperature, and nutritional conditions in a range of various environments. The six isolated strains were found to grow within a pH range of 5.0–9.0, with optimum growth at pH 8.0 (Fig. 1A). The temperature range found suitable for growth of the isolated strains was 20–40 ◦ C, with the optimum being at 30 ◦ C (Fig. 1B). These physiological conditions are reported as the optimum for most of the functions in a living organism. The survivability of the isolated strains following starvation for 35 days showed a similar trend (Fig. 1C); that is, decreasing when no substrate was supplied to the YA broth. 3.2. Urease activity and calcite production Urease is a key enzyme that leads to calcite precipitation and is produced in significant amounts in media containing urea and calcium sources (Stocks-Fischer et al., 1999; Muynck et al., 2010). Among the isolates, strain B-21 showed the highest urease activity (25.4 mM), followed by B-22 (20.0 mM), KJ-64 (10.2 mM), WJ-2 (3.8 mM), KJ-47 (3.7 mM), and KJ-46 (3.3 mM) (Fig. 2). The highest calcite production was observed for strain KJ-64 (17.9 mg/mL), followed by B-22 (17.0 mg/mL), WJ-2 (14.0 mg/mL), KJ-46 (13.6 mg/mL), B-21 (12.5 mg/mL), and KJ-47 (10.5 mg/mL). Urease-producing bacteria can promote calcium carbonate precipitation by hydrolyzing urea and producing ammonium and bicarbonate ions, thereby increasing the pH and accelerating chemical reactions (Achal and Pan, 2011).

Fig. 3. Identification of isolated strains by the polymerase chain reaction (PCR). The PCR products were analyzed by 1.5% agarose gel electrophoresis. Lanes: M, 100 bp ladder; 1, Sporosarcina pasteurii WJ-2; 2, Viridibacillus arenosi B-21; 3, S. soli B-22; 4, Enterobacter cloacae KJ-46; 5, E. cloacae KJ-47; 6, Lysinibacillus sphaericus KJ-64.

largest of the genes coding for the functional subunits of urease and it contains several regions of highly conserved sequences that are suitable as PCR priming sites. Bacterial ureC sequences that might be present in various environments have been selected for ureahydrolysis-based bioremediation research (Gresham et al., 2007). 3.4. Determination of MTC against heavy metals The heavy metal resistance of the isolated strains was tested to investigate their applicability as bioremediators for polluted areas. The results showed that strains KJ-46, KJ-47, and KJ-64 had the highest MTC values for CuSO4 (3000 mg/L), CdCl2 (3000 mg/L), BaCl2 (3000 mg/L), PbCl2 (3000 mg/L), SrCl2 (3000 mg/L), and ZnSO4 (3000 mg/L), whereas strain B-22 has highest value for PbCl2 (3000 mg/L) and SrCl2 (3000 mg/L) (Table 2).

3.3. Molecular analysis of the urease gene A primer pair (ureC-F and ureC-R) was designed to specifically recognize the ureC sequence (Gresham et al., 2007). When this PCR primer pair was tested against all six isolated strains (Fig. 3), 323 bp amplicons were detected in all of them. In this study, all six isolates were positive for the urease test (Table 1). The ureC gene is the Table 2 Maximum tolerance concentrations of isolated strains. Strains

B-21 B-22 KJ-46 KJ-47 KJ-64 W-2

Heavy metals (mg/L) CoCl2

CoSO4

CuSO4

CuCl2

FeCl3

FeSO4

CdCl2

BaCl2

PbCl2

SrCl2

ZnSO4

1000 300 300 1000 1000 3000

1000 300 300 3000 1000 3000

2000 300 3000 3000 3000 300

1000 300 1000 1000 300 300

1000 300 1000 1000 1000 300

2000 300 3000 2000 3000 300

2000 300 3000 3000 3000 300

2000 2000 3000 3000 3000 3000

3000 3000 3000 3000 3000 3000

3000 3000 3000 3000 3000 3000

2000 300 3000 3000 3000 300

Table 3 Antibiotic sensitivity and resistant activity of isolated strains. Antibiotics (␮g/disk)

Chloramphenicol (30) Sulphamethoxazole-trimethoprim (1.25/23.75) Tetracyclin (30) Cephalothin (30) Gentamicin (10) Erythromycin (15) Vancomycin (30) Ampicillin (10) Rifampicin (5) Ciprofloxacin (5) Cefotaxime (30) Cefepime (30) Cefotetan (30) Nalidixic acid (30) Kanamycin (30)

Isolated strains B-21

B-22

KJ-46

KJ-47

KJ-64

W-2

S S S S R S S S S R S S R R R

S S S S S S S S R S S S S S S

S S S R S R R R R S S S S S S

S S S R S R R R R S S S S S S

S S S S S R R R R S S S S S S

S R S S S R S S R S S S S R R

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Fig. 4. Influence of bacterial treatment on the compressive strength of and rate of bacterial survivability in heavy metal cylinder specimens.

Fig. 5. Heavy metal concentrations in the cylinders specimens with bacterial species and without (control).

3.5. Antibiotic resistance pattern of the heavy–metal-resistant strains To further characterize the isolated strains, their susceptibility to various antibiotics was tested (Table 3). Strains KJ-46 and KJ-47 were found to show resistance to both heavy metals and various antibiotics (CF, E, VA, AM, and RA). Many researchers have reported isolation of heavy metal-resistant organisms that showed multidrug resistance to antibiotics such as AM, TE, CIP, GM, E, C, and NA (Kannan and Lee, 2008; Mgbemena et al., 2012; Selvi et al., 2012). It is possible that these bacteria use similar mechanisms to survive under metal- and antibiotic-stressed conditions. Many researchers believe that the combination of antibiotic and metal resistance may not be a fortuitous phenomenon (Calomiris et al., 1984), but rather that bacterial resistance against heavy metals could be directly related to the presence of these elements as environmental contaminants (Raja et al., 2006).

3.6. Heavy metal cylinder specimens In this study, the heavy metal cylinder contained heavy metals, sand, and one of the selected bacterial cultures (KJ-46 or KJ-47); there were no bacterial cells added in control specimens. A compressive strength test was done in order to determine the effectiveness of microbial consolidation by MICP (Fig. 4). Strains KJ-46 and KJ-47 showed compressive strength of 3.5 ± 0.1 MPa and 4.7 ± 0.1 MPa, respectively, after 7 days curing and 7.9 ± 0.1 MPa and 9.3 ± 0.1 MPa, respectively, after 28 days curing. The survivability of the selected strains over a period of 28 days showed a similar trend, decreasing slowly during the first 21 days and then sharply declining thereafter. After incubating the heavy metal cylinder specimens for 28 days, the concentration of heavy metals (Cu and Zn) was estimated from the bacterially prepared specimens and compared with the controls (Fig. 5). The control specimens contained 27.2 mg/kg of Zn, 2.6 mg/kg of Cu, and 785.7 mg/kg of Ca. However, the heavy met-

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Fig. 6. X-Ray diffraction spectra, confirming calcite precipitation induced by Enterobacter cloacae KJ-46 (B) and E. cloacae KJ-47 (C) compared with the control (A). Abbreviations: C, calcite; D, copper squarate; S, quartz; Z, zinc hydroxide.

als and Ca concentrations were significantly high (p < 0.05) in the bacterial specimens. Such results imply the possibility of calcite precipitation, based on MICP, by strains KJ-46 and KJ-47. Heavy metals were found to be preferentially incorporated into the calcite surface during crystal growth (Tang et al., 2007). To confirm the role of MICP in immobilization, we analyzed the immobilized heavy metal cylinder samples by using XRD (Fig. 6). The presence of sharp peaks in the XRD spectra indicated that calcite (CaCO3 ) crystals, copper squarate (C4 CuO4 ), and zinc hydroxide (Zn(OH)2 ) constituted the predominant minerals in the heavy metal cylinder samples, whereas quartz (SiO2 ) crystals was the predominant mineral in the control cylinder samples. The quartz peaks in the control samples may have been derived from the sand.

4. Discussion This investigation highlights the presence of heavy metal ions in the sand sample studied and shows the prevalent occurrence of heavy metal tolerant microbial population in abandoned mine, Korea. Six bacterial strains were used in this study because of their abilities to induce urease activity, produce calcite, and resist heavy metals (Kang et al., 2014, 2015). Several authors pointed out that heavy metal pollution of aquatic and soil ecosystems decreased the microbial diversity (Ezzouhri et al., 2009; Kavamura and Esposito, 2010). Since the pH, temperature, and starvation have roles in enzymatic function as well as overall metabolic efficiency (Fig. 1), these factors do have an effect on survivability. The pH, temperature, and

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starvation profiles of the isolated strains indicated that they have the ability to survive under adverse conditions. In a stressed environment, resistance mechanisms develop in the organism in order for it to survive (Margesin and Schinner, 2001) and bacteria play an important role in the cycling of toxic metals in the biosphere. The introduction of heavy metals in various forms into the environment can considerably modify the microbial communities and affect their activities for their survivability (Karbasizaed et al., 2004). The strains KJ-46, KJ-47, and KJ-64 were able to grow at high concentrations of Cu, Cd, Ba, Pb, Sr and Zn in liquid media (Table 2), which might be important for the capacity of these bacteria to survive in different sources of pollution with elevated heavy metal levels. This varying response of the strains might be due to the differences in their cell wall composition or to variations in their resistance mechanisms (Murthy et al., 2012). Generally, the activity of microbial cells grown at high metal concentrations has been found to be coupled with a variety of specific mechanisms of resistance and environmental factors (Gadd, 2000). The heavy metal resistance mechanisms are sometimes encoded in plasmid genes, facilitating the transfer of the toxic metal resistance factor from one cell to another (Trajanovska et al., 1997). The heavy-metalresistant organisms could be potential agents for bioremediation of environments polluted by heavy metals. In this study, selected strains (KJ-46 and KJ-47) were found to show resistance to both heavy metals and various antibiotics (Table 3). In the last decade, a number of studies have reported that antibiotic-resistant bacteria may arise in the environment through co- or cross-resistance to heavy metals or co-regulation of resistance pathways (Berg et al., 2005; Akinbowale et al., 2007). As the heavy metal and antibiotic resistance genes are often found on the same mobile genetic element, metal pollution can promote the emergence of antibiotic resistances in exposed organisms, which also causes a growing concern in natural and clinical settings (Knapp et al., 2011). Current advances in microbial genomics, physiology and biochemistry could provide the basis for the precise determination of important processes involved in metal–antibiotic resistance interactions. Areas of particular interest include the multifunctional properties of co-resistance determinants and the relative contributions of these resistance systems to the fitness of bacteria in different environmental and clinical settings It is necessary to evaluate potential mechanisms at several levels of biological organization to comprehensively assess the role of metal contaminants as a selective force in maintaining and propagating the pool of antibiotic-resistance determinants in the environment. Since most heavy metal–microbe interactions are initiated at the level of uptake, the uptake mechanism is likely to be closely linked to the mechanism of heavy metal resistance in the microorganism. In this study, the heavy metal cylinder contained heavy metals, sand, and one of the selected bacterial cultures (KJ-46 or KJ-47). A compressive strength test was done in order to determine the effectiveness of microbial consolidation by MICP (Fig. 4). The improvement in compressive strength by the selected strains is due to the deposition of CaCO3 on the cell surfaces and within the pores of the heavy metal–sand matrix, which plugs the pores within the whole cylinder specimen (Ramachandran et al., 2001; Ghosh et al., 2005; Achal et al., 2009; Kang et al., 2016). Bacterial surfaces play an important role in calcium precipitation (Fortin et al., 1997). Commonly, carbonate precipitates develop on the external surface of bacterial cells by successive stratification (Castanier et al., 1999) and bacteria can be embedded within the growing carbonate crystals (Rivadeneyra et al., 1998; Castanier et al., 1999). Rodriguez-Navarro et al. have recently reported that once supersaturation of calcium ions is achieved in the surrounding of bacterial cells and within bacterial biofilms, CaCO3 formation by heterogeneous nucleation occurs readily on bacterial cell walls as well on extracellular polymeric substance (EPS). The heavy metals in cal-

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cium carbonate structures were resistant to gaseous reductants or solution-phase extractants (Hua et al., 2007), implying the longterm stability of heavy metals incorporated in calcium carbonate. As the organisms with a combination of heavy metal and antibiotic resistance would be useful for bioremediation of environments polluted with heavy metals, this study demonstrated the usefulness of heavy metal and antibiotic resistance strains for immobilizing heavy metals in contaminated mine waste to prevent leaching to the surrounding environment. Importantly, our results have demonstrated that the beneficial effects of treatment with selected strains occur within a short period of time. The results of our study suggested that the selected strains might be potential candidates for the development of a biological treatment technology to stabilize heavy metals in abandoned mines. 5. Conclusion Many abandoned mining sites are highly contaminated with multiple types of heavy metals. This study therefore specifically focused on isolating strains with resistance to heavy metals, with an aim to finding a solution to immobilize these pollutants in the environment. Antibiotic-resistant ureolytic bacteria are more frequently associated and strongly correlated with heavy metal resistance. This would be a survival strategy for these organisms to proliferate ahead of other microorganisms in the environment, since most of the possible antibiosis that could occur in such sites would not affect them. This could be a very important feature, as reported by Sarmah et al. (2006), as the antibiotic concentrations in some environments have been found to be high enough to inhibit many bacterial species. Additionally, we demonstrated that ureolytic bacteria can be used for the immobilization of heavy metals by MICP. The metabolically active system of heavy metal resistance by these isolates is novel and represents a point of interest for possible environmental applications to immobilize toxic heavy metals from contaminated environments. Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2015R1D1A1A01056788). References Achal, V., Pan, X., 2011. Characterization of urease and carbonic anhydrase producing bacteria and their role in calcite precipitation. Curr. Microbiol. 62, 894–902. Achal, V., Mukherjee, A., Basu, P.C., Reddy, M.S., 2009. Strain improvement of Sporosarcina pasteurii for enhanced urease and calcite production. J. Ind. Microbiol. Biotechnol. 36, 981–988. Akinbowale, O.L., Peng, H., Grant, P., Barton, M.D., 2007. Antibiotic and heavy metal resistance in motile aeromonads and pseudomonads from rainbow trout (Oncorhynchus mykiss) farms in Australia. Int. J. Antimicrob. Agents 30, 177–182. Alonso, A., Sanchez, P., Martinez, J.L., 2000. Stenotrophomonas maltophilia D457R contains a cluster of genes from gram-positive bacteria involved in antibiotic and heavy-metal resistance. Antimicrob. Agents Chemother. 44, 1778–1782. Anbu, P., Kang, C.H., Shin, Y.J., So, J.S., 2016. Formations of calcium carbonate minerals by bacteria and its multiple applications. Springerplus 5, 250–276. Avery, S.V., 1995. Cesium accumulation by microorganisms: uptake mechanisms, cation competition, compactmentalisation and toxicity. J. Ind. Microbiol. 14, 76–84. Bachate, S.P., Nandre, V.S., Ghatpande, N.S., Kodam, K.M., 2013. Simultaneous reduction of Cr(VI) and oxidation of As(III) by Bacillus firmus TE7 isolated from tannery effluent. Chemosphere 90 (8), 2273–2278. Berg, J., Tom-Petersen, A., Nybroe, O., 2005. Copper amendment of agricultural soil selects for bacterial antibiotic resistance in the field. Lett. Appl. Microbiol. 40, 146–151. CLSI, 2006. Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria; Approved Guideline M45-A. Clinical and Laboratory Standards Institute, Waune, PA.

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