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Bioremediation of heavy metals by using bacterial mixtures
Ecological Engineering 89 (2016) 64–69
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Ecological Engineering journal homepage: www.elsevier.com/locate/ecoleng
Bioremediation of heavy metals by using bacterial mixtures Chang-Ho Kang a , Yoon-Jung Kwon b , Jae-Seong So a,∗ a b
Department of Biological Engineering, Inha University, Incheon 22212, South Korea Department of Organic and Nano System Engineering, Konkuk University, Seoul 05029, Korea
a r t i c l e
i n f o
Article history: Received 25 March 2015 Received in revised form 4 November 2015 Accepted 26 January 2016 Keywords: MICP Bioremediation Bacterial mixtures Urease Heavy metal
a b s t r a c t Environmental pollution by heavy and toxic metals because of mining, metallurgic processes, and other chemical industries is a worldwide problem affecting both human health and the environment. The aim of this study was to investigate the synergistic effect of bacterial mixtures on the bioremediation of a mixture of Pb, Cd, and Cu from contaminated soils. Compared to the single culture method, the bacterial mixtures showed higher growth rate, urease activity, and resistance to heavy metals. Four bacterial strains were isolated and identified from bacterial mixtures—Viridibacillus arenosi B-21, Sporosarcina soli B-22, Enterobacter cloacae KJ-46, and E. cloacae KJ-47, which obtained from an abandoned mine site in Korea and showed effective microbially induced calcite precipitation (MICP). The following parameters were monitored during the course of the experiment: optical density, pH, urease activity, calcite production, tolreance to heavy metals, and impermeability test. Synergistic effects on the remediation of various heavy metals via modification of the bacterial mixtures were observed and, after 48 h, remediation of 98.3% for Pb, 85.4% for Cd, and 5.6% for Cu were recorded. Compared with single strain cultures, the bacterial mixtures demonstrated greater resistance and efficiency for the remediation of heavy metals. Thus, our results show that the use of bacterial mixtures is useful in the bioremediation of heavy metals from the contaminated environment. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The accumulation of heavy metals in water, sediments, and soils has led to serious environmental problems. In recent years, several processes have been developed with the aim of reducing or recovering heavy metals from contaminated environments (Akinci and Guven, 2011). Physical and chemical approaches are capable of removing a broad spectrum of contaminants, but the main disadvantages of these methods lie in the increased energy consumption and the need of additional chemicals (Ilhan et al., 2004). In recent years, the processes such as bioleaching, biosorption, and bioprecipitation are all based on the use of microorganisms that have the ability to solubilize, adsorb, or precipitate heavy metals (Ballester et al., 1992; Zouboulis et al., 1997). To date, most of the research on microbially induced calcite precipitation (MICP) has been confined to ureolytic bacteria, with specific focus on the catalysis of urea hydrolysis (Ferris et al., 2003), efficiency of calcite production (Muynck et al., 2010; van Paassen et al., 2010), and modification of soil physical properties by model bacteria (Burbank et al., 2011; De Jong
∗ Corresponding author. Tel.: +82 32 860 8666; fax: +82 32 872 4046. E-mail address: [email protected] (J.-S. So). http://dx.doi.org/10.1016/j.ecoleng.2016.01.023 0925-8574/© 2016 Elsevier B.V. All rights reserved.
et al., 2010). The MICP has been shown to increase the shear strength of porous materials (De Jong et al., 2006; Harkes et al., 2010). MICP arises when the following reaction catalyzed by urease: (NH2 )2 CO + 2H2 O → 2NH4+ + CO3 2− occurs in the presence of dissolved calcium ions, leading to the precipitation of calcium carbonate crystal: Ca2+ + CO3 2− → CaCO3 (s). The crystals formed by this process create bridges between particles, thus improving the strength and stiffness of the material (Harkes et al., 2010). Urease-induced CaCO3 can fill pore spaces within various soil matrices and cement soil grains together to form sandstone (Burbank et al., 2011; Deepak et al., 2009; De Jong et al., 2006). Precipitation of CaCO3 induced by the urease-catalyzed hydrolysis of urea has been shown to change the engineering properties of geomaterials (Burbank et al., 2011; Whiffin et al., 2007). Bacterial mixtures systems have long been used to study the interactions between cell populations and fundamental cell–cell interactions. Recently, these systems have been of particular interest to synthetic biologists for the study and engineering of complex multicellular synthetic systems. At the basic level, a co-culture is a cell culture set-up, in which two or more populations of cells are grown with some degree of contact between them (Goers et al., 2014). The ultimate aim of the bacterial mixtures system is to deliver societal benefits via its industrial, medical, and environmental applications (Chen, 2012; Kitney and Freemont, 2012).
C.-H. Kang et al. / Ecological Engineering 89 (2016) 64–69
Therefore, many bacterial mixtures systems are developed for future industrial, medical, or environmental applications. Although the synergetic interactions between metals and ureolytic bacteria have attracted a fair share of attention, the effects of metal contaminated environments on bacterial mixtures growth are still unknown. The utilization of microorganisms with proven remediation potential and survivability in the contaminated environment is crucial for a successful bioremediation. In view of this, the present paper aims to study the remediation capacity of heavy metals by pure and mixed bacterial cultures, for bioremediation process applications. 2. Materials and methods
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350 mM calcium chloride dihydrate solution and the mixture was centrifuged at 16,179 × g for 5 min at 25 ◦ C to collect the precipitate. Finally, the precipitate was dried at 50 ◦ C for 48 h prior to being weighed. 2.4. Determination of the minimum inhibitory concentration The tolerance experiments were carried out in 250 mL conical flasks, each containing 45 mL of YA broth. Approximately equal numbers of cells from the four isolated strains were used and the bacterial mixtures with an initial cell density of 1 × 108 cells/mL for each of the isolated strains. All cultures were incubated at 30 ◦ C and agitated at 200 rpm. To study the influence of heavy metals on bacterial growth, bacterial mixtures were exposed to a mix of heavy metals with individual concentrations ranging from 0 to 10 mM.
2.1. Microorganisms and culture conditions 2.5. Impermeability test Four bacterial strains were isolated from an abandoned mine soil in our previous work (Kang et al., 2015). These strains include Viridibacillus arenosi B-21 (B-21), Sporosarcina soli B-22 (B-22), Enterobacter cloacae KJ-46 (KJ-46), and E. cloacae KJ-47 (KJ-47). Three sets of experiments were conducted under aerobic conditions using defined co-culture—A1, mixed bacterial cultures of B-21 and B-22; A2, mixed bacterial cultures of KJ-46 and KJ-47; A3, mixed bacterial cultures of B-21, B-22, KJ-46, and KJ-47. These strains were pre-selected based on their high levels of urease activity and calcite production. Also, these strains had the highest heavy metal tolerance. Their sequences have been deposited in GenBank with accession numbers KJ671467 (B-21), KJ485701 (B-22), KF598853 (KJ-46), and KF598854 (KJ-47). The bacterial mixtures were routinely grown at 30 ◦ C in YA (yeast extract 20 g/L and ammonium sulfate 10 g/L at pH7) broth. The final pH of the medium was adjusted to 7.0.
The single cultures and bacterial mixtures grown in YA broth overnight were harvested by centrifugation at 6000 × g for 5 min, washed twice, and resuspended in a 0.9% sodium chloride solution at a final OD600 of 1.0 (equivalent to 1 × 108 cells/mL). Sterile silica sand (200 g, 0.45–0.7 mm, Joomoonjin Sand Co. Ltd., Korea) was mixed with 10 mL urea (40 g/L) and calcium chloride dihydrate solution (25 g/L). The sand slurry (40 g) was packed into a 25 mL plastic column (Corning Co. Ltd., Corning, NY, USA). After being dried for 48 h at 50 ◦ C, the columns were run once by gravity with 10 mL of cell suspension. The columns were stored for 48 h to allow calcite crystal growth. Then, 2 mL of crystal violet (CV) was pipetted onto the packed sand column. The degree of impermeability was determined by measuring the migration distance of CV. 2.6. Bioremediation of heavy metal
2.2. Preparation of heavy metal stock solutions For the preparation of 1 M mixtures of heavy metal stock solutions the necessary quantities of: CdCl2 ·5H2 O (Kanto Chemical Co., Ltd., Tokyo, Japan), PbCl2 (Junsei Chemical Co., Ltd., Tokyo, Japan), and CuCl2 (Samchun Pure Chemical Co., Ltd., Korea) were dissolved in Milli-Q water. All solutions were filtered through a 0.22 m filter (Pall Co., MI, USA). Working concentrations of heavy metal mixture were obtained by serial dilutions. The stock solutions were stored in the dark at 4 ◦ C. 2.3. Measurement of urease activity and calcite production Experimental conditions were the same as for the bacterial mixtures growth described above. To establish the bacterial mixtures, first, a bacterial culture was added to the experimental flasks and incubated at 30 ◦ C for 24 h with continuous agitation at 200 rpm. Urease activity was determined using the phenol-hypochlorite assay (Natarajan, 1995). The bacterial suspension (250 L) was added to 250 L of 0.1 M sodium phosphate buffer containing 500 L of 3 M urea solution. The mixture was incubated at 37 ◦ C with regular time intervals. Subsequently, 2 mL of phenol nitroprusside solution were added to an alkaline hypochlorite solution and, then, incubated at 50 ◦ C for 40 min. After incubation, absorbance was measured at 626 nm with ammonium chloride (0–10 M) as a standard. One unit of urease activity was defined as the amount of enzyme that catalyzed the hydrolysis of 1 M urea per min. To produce calcite, the isolates were cultured in YA broth for 24 h and subcultured onto BPU broth (3 g/L beef extract, 5 g/L peptone, and 20 g/L urea at pH 7) at 30 ◦ C for 72 h with continuous agitation at 200 rpm. The bacterial suspension (500 L) was added to 500 L of
Five milliliters of the overnight pre-culture were inoculated into 45 mL of YA broth contained in 250 mL conical flasks. The YA broth contained 5 mM urea and 25 mM CaCl2 , which were filter sterilized using a 0.22 m filter, and was supplemented with 2 mM of heavy metal mixture. The flasks were incubated in a shaking incubator (200 rpm) at 30 ◦ C for 48 h. Control sets without any added bacterial cells were also included in all experiments. After incubation, the cultures were centrifuged at 8000 × g for 15 min. The heavy metal bioremediation by single cultures and bacterial mixtures (A3) were calculated as the percentage difference between the initial and final concentrations of heavy metals present in the supernatants. Concentrations of heavy metals were measured using a Perkin-Elmer OPTIMA-7300DV inductively coupled plasma optical spectrometer (ICP-OES, PerkinElmer, Inc., USA). 2.7. Column test The silica sand samples were sieved, washed, air-dried, and sterilized before the experiments. The pH of the sand suspended in distilled water was 8.5. The heavy metal bioremediation studies were performed in 50 mm diameter and 500 mm length columns (OMG Chemical Co., Korea), containing 1.0 kg silica sand supplemented with 2 mM mixture of heavy metals. The heavy metals were thoroughly mixed into the sand while in solution form. The bacterial mixtures culture (A3, equivalent to 108 CFU/mL) was grown overnight in YA broth. The control treatment consisted of the same set-up as the experimental treatment but without the addition of bacterial cells. Bioremediation was determined by measuring the time required for 100 mL of 5 mM urea and 25 mM calcium chloride to pass through the column. The experiment was performed at room temperature (25 ◦ C) and run for 3 d. After the experiment
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Fig. 1. Urease activity and calcite production of single strains and bacterial mixtures (values are means ± SD).
ended, the effluent samples were collected from the bottom of the column with 100 mL of distilled water. The concentrations of heavy metals in the effluent samples were determined by ICP-OES.
The results were expressed as the mean ± S.D. of three experiments. Statistical analysis was performed by the Student’s t-test, and a P value of <0.05 was considered to indicate a significant difference.
environments than a single culture (Brenner et al., 2008). Bacterial resistance to heavy metals is an important factor to be considered in the study of remediation because it is directly related to the survival and growth of the bacteria being used to recover contaminated sites (Li and Ramakrishna, 2011). It is known that metal resistance systems in bacteria are abundant and widespread, and their appearance frequencies range from a small percentage of the isolates in clean environments to nearly all isolates in heavily polluted environments (Barkay and Schaefer, 2001; Zouboulis et al., 2004).
3. Results and discussion
3.3. Impermeability test
3.1. Urease activity and calcite production
In this study, the bacterial mixtures containing all strains showed a reduced CV migration length compared with that of single cultures (Fig. 3). The advantages of the impermeability test are its simplicity of operation, lower cost compared with other processes, and the absence of calcite formation (Kang et al., 2014). The reduction in the retention time can be attributed to the calcium carbonate crystals that were deposited between sand particles, which resulted in plugging. Improvement in the strength of sand columns due to bacterially induced calcium carbonate has also been previously reported (De Jong et al., 2010; Whiffin et al., 2007). Recent research initiatives have shown that calcite crystals form cohesive “bridges” between existing sand grains, increasing the stiffness of the sand while causing only a limited decrease in permeability (De Jong et al., 2010; van Paassen et al., 2010).
2.8. Statistical analysis
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). All isolated strains produced amounts of urease. Among the isolates, the bacterial mixtures showed the highest levels of urease activity (29.2 mM), followed by KJ-47 (27.5 mM), KJ-46 (24.7 mM), B-21 (24.7 mM), and B-22 (21.7 mM) (Fig. 1). The highest calcite production was observed in KJ-47 (17.6 mg/mL), followed by B-22 (17.2 mg/mL), bacterial mixtures (A3, 15.0 mg/mL), KJ-46 (14.0 mg/mL), and B-21 (12.5 mg/mL). Compared with the single culture, the bacterial mixtures has many advantages because urea hydrolysis rates were higher in it and the pH values of the bacterial mixtures increased more rapidly than those of the different single cultures (data not shown). 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). 3.2. Heavy metal tolerance study The bacterial mixtures containing all strains tolerated the highest minimum inhibitory concentration (MIC) of heavy metals, 9 mM (Fig. 2). Among the bacterial strains and mixtures tested, three of them (A3, KJ-46, and KJ-47) tolerated a heavy metal MIC of 6 mM. Previous research implied that because the members of bacterial mixtures communicate and differentiate, the bacterial mixtures can perform more complex tasks and survive in more changeable
3.4. Culture growth patterns and remediation by bacterial mixtures The microbial growth profiles during the flask-scale experiments using different bacterial mixtures designs are shown in Fig. 4. Different growth patterns were observed for different bacterial mixtures designs (A1 and A2). Over time pH changes are shown in Fig. 4A. Urea hydrolysis is expected to lead to a pH increase in the medium due to ammonium production. Initial pH values for all treatments were approximately 6.5, probably due to the YA broth pH. In the experiment with different bacterial mixtures, pH values increased rapidly for 72 h (by approximately pH 2.5) until they reached at an approximate average pH of 8.6. A similar phenomenon was also described by Tobler et al. (2011)
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Fig. 2. Minimum inhibitory concentration of a mixture of heavy metals on single strains and bacterial mixtures (values are means ± SD).
during the induction of urea hydrolysis in bacterial mixtures of indigenous soil bacteria. During absorbance measurements, the stationary growth phase started 72 h after the start of the experiment (Fig. 4B), whereas during CFU measurements, the death phase started 48 h after the start of the experiment (Fig. 4C). In our experiment, the pH values increased rapidly for 48 h until they reached at an approximate pH value of 8.22 (Fig. 5A) because urea hydrolysis rates are higher in bacterial mixtures. However, during the CFU measurement, the death phase started 48 h after the start of the experiment (Fig. 5A). The removal of heavy metals by the bacterial mixtures was assessed using a 2 mM mixture of heavy metals, and the remediation potentials of the bacterial mixtures were observed to vary between 5.56% and 98.25% (Fig. 5B). Pb, Cd, and Cu concentrations were reduced to 0.06, 19.34, and 137.50 mg/L in about 48 h, respectively. This amounted to about 98.25%, 85.39%, and 5.56% Pb, Cd, and Cu removal by bioremediation, respectively. As previous studies have shown that
precipitated CaCO3 encapsulates bacteria cells (Mitchell and Ferris, 2006; Cuthbert et al., 2012), it is assumed that the electronegativity of the bacterial cell wall favors the adsorption of cations such as calcium ions, thus, facilitating the process of CaCO3 precipitation on the cell wall (Schultze-Lam et al., 1996). Various studies have also reported that CaCO3 may be the dominant sorbent for a variety of metals in carbonate aquifers (Zachara et al., 1991). Furthermore, heavy metals were reported to be sorbed on the surface of calcite (Lorens, 1981), with sorption being defined as a surface process irrespective of mechanism, adsorption, or precipitation (Sposito, 1984). 3.5. Column tests Bioremediation rates can be estimated based on the results from Fig. 6. The results showed that bioremediation rates increased in the following order: Cd (42.4%) < Cu (67.2%) < Pb (98.5%).
Fig. 3. Migration length of crystal violet under single strains and different bacterial mixtures (values are means ± SD). * Indicates P < 0.05.
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Fig. 5. Time dependency of growth pattern and heavy metal removal capacity of the bacterial mixtures.
Fig. 4. Changes over time in pH (A), OD at 600 nm (B), and colony forming unit (C) for treatments using different bacterial mixtures designs.
Adsorption affinity of heavy metals onto bacteria may be related to the electronegativity of metal ions as described by other studies (Merdy et al., 2009; Seco et al., 1997), which reported that attraction between metals and bacteria is higher with greater electronegativity. The selectivity in both single and multi-component solutions is higher for Pb2+ than for Cu2+ . In addition, the high ionic radius of ´˚ must induce a quick saturation of adsorption sites due Pb2+ (1.12 A) ´˚ These facts could to steric hindrance compared with Cu2+ (0.70 A). explain the reactivity of Cu and Pb with bacteria surface sites. Pb2+ adsorbs onto calcite and, thus, the Pb2+ ions move into Ca2+ sites, despite the large ionic radius of Pb2+ relative to Ca2+ (Sturchio et al., 1997). To further confirm the role of MICP in heavy metal bioremediation, remediated sand samples were analyzed by X-ray diffraction (XRD). XRD spectra indicated the presence of cerussite (lead carbonate, PbCO3 ), otavite (cadmium carbonate, CdCO3 ), and copper carbonate (CuCO3 ) crystals as the predominant minerals, as evidenced by the detection of sharp peaks in analysis of bioremediated samples, in contrast to the control, where peaks corresponding to quartz (SiO2 ) represented the sharpest peaks (Fig. 7).
Fig. 6. Removal of heavy metals by the bacterial mixtures (values are means ± SD).
The quartz peaks in the control samples might be attributed to the sand sample. A mixture of heavy metals was found to co-precipitate with CaCO3 as cerussite (lead carbonate, PbCO3 ) in the bioremediation experiments, while otavite (cadmium carbonate, CdCO3 ) and copper carbonate (CuCO3 ) peaks were also detected. The overall reactions involved in the bioremediation process include: production of NH4 + and HCO3 − by urease, desorption of Ca2+ and/or heavy metal ions from solid surfaces by the NH4 + and HCO3 − promoted precipitation of CaCO3 , and the co-precipitation of heavy metals.
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Fig. 7. XRD spectra, confirming the bioremediation of heavy metals by the bacterial mixtures grown in a sand column; XRD pattern of the control sand column (A) and bioremediated sand column (B). Identified peaks: C, CuCO3 ; O, otavite (CdCO3 ); P, cerussite (PbCO3 ); Q, quartz (SiO2 ).
4. Conclusion In this study, isolated strains were used to evaluate the synergistic effects of bacterial mixtures on bioremediation efficiency. At the end of the experimental period, the bacterial mixtures were found to be the most effective bacterial method, in comparison to individual culture methods. Compared with single strain cultures, the bacterial mixtures showed higher growth rate, urease activity, and resistance to heavy metals. Also, we demonstrated that the bacterial mixtures exhibited a considerably higher heavy metal bioremediation capacity than individual cultures, which might due to higher bacterial cell density at high levels of heavy metals. According to the column test results, the highest bioremediation efficiencies were noted for Pb, while the lowest removal efficiencies were detected for Cd. Bioremediation with the bacterial mixtures of isolated strains is an effective method for heavy metal removal from contaminated environments. This may prove to be a good strategy for developing an effective, efficient, and economic method of heavy metal bioremediation, and the introduction of these indigenous bacteria could be used for contaminated soil bioremediation without disturbing the target environment. 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. Akinci, G., Guven, D.E., 2011. Bioleaching of heavy metals contaminated sediment by pure and mixed cultures of Acidithiobacillus spp. Desalination 268, 221–226.
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Ecological Engineering journal homepage: www.elsevier.com/locate/ecoleng
Bioremediation of heavy metals by using bacterial mixtures Chang-Ho Kang a , Yoon-Jung Kwon b , Jae-Seong So a,∗ a b
Department of Biological Engineering, Inha University, Incheon 22212, South Korea Department of Organic and Nano System Engineering, Konkuk University, Seoul 05029, Korea
a r t i c l e
i n f o
Article history: Received 25 March 2015 Received in revised form 4 November 2015 Accepted 26 January 2016 Keywords: MICP Bioremediation Bacterial mixtures Urease Heavy metal
a b s t r a c t Environmental pollution by heavy and toxic metals because of mining, metallurgic processes, and other chemical industries is a worldwide problem affecting both human health and the environment. The aim of this study was to investigate the synergistic effect of bacterial mixtures on the bioremediation of a mixture of Pb, Cd, and Cu from contaminated soils. Compared to the single culture method, the bacterial mixtures showed higher growth rate, urease activity, and resistance to heavy metals. Four bacterial strains were isolated and identified from bacterial mixtures—Viridibacillus arenosi B-21, Sporosarcina soli B-22, Enterobacter cloacae KJ-46, and E. cloacae KJ-47, which obtained from an abandoned mine site in Korea and showed effective microbially induced calcite precipitation (MICP). The following parameters were monitored during the course of the experiment: optical density, pH, urease activity, calcite production, tolreance to heavy metals, and impermeability test. Synergistic effects on the remediation of various heavy metals via modification of the bacterial mixtures were observed and, after 48 h, remediation of 98.3% for Pb, 85.4% for Cd, and 5.6% for Cu were recorded. Compared with single strain cultures, the bacterial mixtures demonstrated greater resistance and efficiency for the remediation of heavy metals. Thus, our results show that the use of bacterial mixtures is useful in the bioremediation of heavy metals from the contaminated environment. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The accumulation of heavy metals in water, sediments, and soils has led to serious environmental problems. In recent years, several processes have been developed with the aim of reducing or recovering heavy metals from contaminated environments (Akinci and Guven, 2011). Physical and chemical approaches are capable of removing a broad spectrum of contaminants, but the main disadvantages of these methods lie in the increased energy consumption and the need of additional chemicals (Ilhan et al., 2004). In recent years, the processes such as bioleaching, biosorption, and bioprecipitation are all based on the use of microorganisms that have the ability to solubilize, adsorb, or precipitate heavy metals (Ballester et al., 1992; Zouboulis et al., 1997). To date, most of the research on microbially induced calcite precipitation (MICP) has been confined to ureolytic bacteria, with specific focus on the catalysis of urea hydrolysis (Ferris et al., 2003), efficiency of calcite production (Muynck et al., 2010; van Paassen et al., 2010), and modification of soil physical properties by model bacteria (Burbank et al., 2011; De Jong
∗ Corresponding author. Tel.: +82 32 860 8666; fax: +82 32 872 4046. E-mail address: [email protected] (J.-S. So). http://dx.doi.org/10.1016/j.ecoleng.2016.01.023 0925-8574/© 2016 Elsevier B.V. All rights reserved.
et al., 2010). The MICP has been shown to increase the shear strength of porous materials (De Jong et al., 2006; Harkes et al., 2010). MICP arises when the following reaction catalyzed by urease: (NH2 )2 CO + 2H2 O → 2NH4+ + CO3 2− occurs in the presence of dissolved calcium ions, leading to the precipitation of calcium carbonate crystal: Ca2+ + CO3 2− → CaCO3 (s). The crystals formed by this process create bridges between particles, thus improving the strength and stiffness of the material (Harkes et al., 2010). Urease-induced CaCO3 can fill pore spaces within various soil matrices and cement soil grains together to form sandstone (Burbank et al., 2011; Deepak et al., 2009; De Jong et al., 2006). Precipitation of CaCO3 induced by the urease-catalyzed hydrolysis of urea has been shown to change the engineering properties of geomaterials (Burbank et al., 2011; Whiffin et al., 2007). Bacterial mixtures systems have long been used to study the interactions between cell populations and fundamental cell–cell interactions. Recently, these systems have been of particular interest to synthetic biologists for the study and engineering of complex multicellular synthetic systems. At the basic level, a co-culture is a cell culture set-up, in which two or more populations of cells are grown with some degree of contact between them (Goers et al., 2014). The ultimate aim of the bacterial mixtures system is to deliver societal benefits via its industrial, medical, and environmental applications (Chen, 2012; Kitney and Freemont, 2012).
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Therefore, many bacterial mixtures systems are developed for future industrial, medical, or environmental applications. Although the synergetic interactions between metals and ureolytic bacteria have attracted a fair share of attention, the effects of metal contaminated environments on bacterial mixtures growth are still unknown. The utilization of microorganisms with proven remediation potential and survivability in the contaminated environment is crucial for a successful bioremediation. In view of this, the present paper aims to study the remediation capacity of heavy metals by pure and mixed bacterial cultures, for bioremediation process applications. 2. Materials and methods
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350 mM calcium chloride dihydrate solution and the mixture was centrifuged at 16,179 × g for 5 min at 25 ◦ C to collect the precipitate. Finally, the precipitate was dried at 50 ◦ C for 48 h prior to being weighed. 2.4. Determination of the minimum inhibitory concentration The tolerance experiments were carried out in 250 mL conical flasks, each containing 45 mL of YA broth. Approximately equal numbers of cells from the four isolated strains were used and the bacterial mixtures with an initial cell density of 1 × 108 cells/mL for each of the isolated strains. All cultures were incubated at 30 ◦ C and agitated at 200 rpm. To study the influence of heavy metals on bacterial growth, bacterial mixtures were exposed to a mix of heavy metals with individual concentrations ranging from 0 to 10 mM.
2.1. Microorganisms and culture conditions 2.5. Impermeability test Four bacterial strains were isolated from an abandoned mine soil in our previous work (Kang et al., 2015). These strains include Viridibacillus arenosi B-21 (B-21), Sporosarcina soli B-22 (B-22), Enterobacter cloacae KJ-46 (KJ-46), and E. cloacae KJ-47 (KJ-47). Three sets of experiments were conducted under aerobic conditions using defined co-culture—A1, mixed bacterial cultures of B-21 and B-22; A2, mixed bacterial cultures of KJ-46 and KJ-47; A3, mixed bacterial cultures of B-21, B-22, KJ-46, and KJ-47. These strains were pre-selected based on their high levels of urease activity and calcite production. Also, these strains had the highest heavy metal tolerance. Their sequences have been deposited in GenBank with accession numbers KJ671467 (B-21), KJ485701 (B-22), KF598853 (KJ-46), and KF598854 (KJ-47). The bacterial mixtures were routinely grown at 30 ◦ C in YA (yeast extract 20 g/L and ammonium sulfate 10 g/L at pH7) broth. The final pH of the medium was adjusted to 7.0.
The single cultures and bacterial mixtures grown in YA broth overnight were harvested by centrifugation at 6000 × g for 5 min, washed twice, and resuspended in a 0.9% sodium chloride solution at a final OD600 of 1.0 (equivalent to 1 × 108 cells/mL). Sterile silica sand (200 g, 0.45–0.7 mm, Joomoonjin Sand Co. Ltd., Korea) was mixed with 10 mL urea (40 g/L) and calcium chloride dihydrate solution (25 g/L). The sand slurry (40 g) was packed into a 25 mL plastic column (Corning Co. Ltd., Corning, NY, USA). After being dried for 48 h at 50 ◦ C, the columns were run once by gravity with 10 mL of cell suspension. The columns were stored for 48 h to allow calcite crystal growth. Then, 2 mL of crystal violet (CV) was pipetted onto the packed sand column. The degree of impermeability was determined by measuring the migration distance of CV. 2.6. Bioremediation of heavy metal
2.2. Preparation of heavy metal stock solutions For the preparation of 1 M mixtures of heavy metal stock solutions the necessary quantities of: CdCl2 ·5H2 O (Kanto Chemical Co., Ltd., Tokyo, Japan), PbCl2 (Junsei Chemical Co., Ltd., Tokyo, Japan), and CuCl2 (Samchun Pure Chemical Co., Ltd., Korea) were dissolved in Milli-Q water. All solutions were filtered through a 0.22 m filter (Pall Co., MI, USA). Working concentrations of heavy metal mixture were obtained by serial dilutions. The stock solutions were stored in the dark at 4 ◦ C. 2.3. Measurement of urease activity and calcite production Experimental conditions were the same as for the bacterial mixtures growth described above. To establish the bacterial mixtures, first, a bacterial culture was added to the experimental flasks and incubated at 30 ◦ C for 24 h with continuous agitation at 200 rpm. Urease activity was determined using the phenol-hypochlorite assay (Natarajan, 1995). The bacterial suspension (250 L) was added to 250 L of 0.1 M sodium phosphate buffer containing 500 L of 3 M urea solution. The mixture was incubated at 37 ◦ C with regular time intervals. Subsequently, 2 mL of phenol nitroprusside solution were added to an alkaline hypochlorite solution and, then, incubated at 50 ◦ C for 40 min. After incubation, absorbance was measured at 626 nm with ammonium chloride (0–10 M) as a standard. One unit of urease activity was defined as the amount of enzyme that catalyzed the hydrolysis of 1 M urea per min. To produce calcite, the isolates were cultured in YA broth for 24 h and subcultured onto BPU broth (3 g/L beef extract, 5 g/L peptone, and 20 g/L urea at pH 7) at 30 ◦ C for 72 h with continuous agitation at 200 rpm. The bacterial suspension (500 L) was added to 500 L of
Five milliliters of the overnight pre-culture were inoculated into 45 mL of YA broth contained in 250 mL conical flasks. The YA broth contained 5 mM urea and 25 mM CaCl2 , which were filter sterilized using a 0.22 m filter, and was supplemented with 2 mM of heavy metal mixture. The flasks were incubated in a shaking incubator (200 rpm) at 30 ◦ C for 48 h. Control sets without any added bacterial cells were also included in all experiments. After incubation, the cultures were centrifuged at 8000 × g for 15 min. The heavy metal bioremediation by single cultures and bacterial mixtures (A3) were calculated as the percentage difference between the initial and final concentrations of heavy metals present in the supernatants. Concentrations of heavy metals were measured using a Perkin-Elmer OPTIMA-7300DV inductively coupled plasma optical spectrometer (ICP-OES, PerkinElmer, Inc., USA). 2.7. Column test The silica sand samples were sieved, washed, air-dried, and sterilized before the experiments. The pH of the sand suspended in distilled water was 8.5. The heavy metal bioremediation studies were performed in 50 mm diameter and 500 mm length columns (OMG Chemical Co., Korea), containing 1.0 kg silica sand supplemented with 2 mM mixture of heavy metals. The heavy metals were thoroughly mixed into the sand while in solution form. The bacterial mixtures culture (A3, equivalent to 108 CFU/mL) was grown overnight in YA broth. The control treatment consisted of the same set-up as the experimental treatment but without the addition of bacterial cells. Bioremediation was determined by measuring the time required for 100 mL of 5 mM urea and 25 mM calcium chloride to pass through the column. The experiment was performed at room temperature (25 ◦ C) and run for 3 d. After the experiment
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Fig. 1. Urease activity and calcite production of single strains and bacterial mixtures (values are means ± SD).
ended, the effluent samples were collected from the bottom of the column with 100 mL of distilled water. The concentrations of heavy metals in the effluent samples were determined by ICP-OES.
The results were expressed as the mean ± S.D. of three experiments. Statistical analysis was performed by the Student’s t-test, and a P value of <0.05 was considered to indicate a significant difference.
environments than a single culture (Brenner et al., 2008). Bacterial resistance to heavy metals is an important factor to be considered in the study of remediation because it is directly related to the survival and growth of the bacteria being used to recover contaminated sites (Li and Ramakrishna, 2011). It is known that metal resistance systems in bacteria are abundant and widespread, and their appearance frequencies range from a small percentage of the isolates in clean environments to nearly all isolates in heavily polluted environments (Barkay and Schaefer, 2001; Zouboulis et al., 2004).
3. Results and discussion
3.3. Impermeability test
3.1. Urease activity and calcite production
In this study, the bacterial mixtures containing all strains showed a reduced CV migration length compared with that of single cultures (Fig. 3). The advantages of the impermeability test are its simplicity of operation, lower cost compared with other processes, and the absence of calcite formation (Kang et al., 2014). The reduction in the retention time can be attributed to the calcium carbonate crystals that were deposited between sand particles, which resulted in plugging. Improvement in the strength of sand columns due to bacterially induced calcium carbonate has also been previously reported (De Jong et al., 2010; Whiffin et al., 2007). Recent research initiatives have shown that calcite crystals form cohesive “bridges” between existing sand grains, increasing the stiffness of the sand while causing only a limited decrease in permeability (De Jong et al., 2010; van Paassen et al., 2010).
2.8. Statistical analysis
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). All isolated strains produced amounts of urease. Among the isolates, the bacterial mixtures showed the highest levels of urease activity (29.2 mM), followed by KJ-47 (27.5 mM), KJ-46 (24.7 mM), B-21 (24.7 mM), and B-22 (21.7 mM) (Fig. 1). The highest calcite production was observed in KJ-47 (17.6 mg/mL), followed by B-22 (17.2 mg/mL), bacterial mixtures (A3, 15.0 mg/mL), KJ-46 (14.0 mg/mL), and B-21 (12.5 mg/mL). Compared with the single culture, the bacterial mixtures has many advantages because urea hydrolysis rates were higher in it and the pH values of the bacterial mixtures increased more rapidly than those of the different single cultures (data not shown). 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). 3.2. Heavy metal tolerance study The bacterial mixtures containing all strains tolerated the highest minimum inhibitory concentration (MIC) of heavy metals, 9 mM (Fig. 2). Among the bacterial strains and mixtures tested, three of them (A3, KJ-46, and KJ-47) tolerated a heavy metal MIC of 6 mM. Previous research implied that because the members of bacterial mixtures communicate and differentiate, the bacterial mixtures can perform more complex tasks and survive in more changeable
3.4. Culture growth patterns and remediation by bacterial mixtures The microbial growth profiles during the flask-scale experiments using different bacterial mixtures designs are shown in Fig. 4. Different growth patterns were observed for different bacterial mixtures designs (A1 and A2). Over time pH changes are shown in Fig. 4A. Urea hydrolysis is expected to lead to a pH increase in the medium due to ammonium production. Initial pH values for all treatments were approximately 6.5, probably due to the YA broth pH. In the experiment with different bacterial mixtures, pH values increased rapidly for 72 h (by approximately pH 2.5) until they reached at an approximate average pH of 8.6. A similar phenomenon was also described by Tobler et al. (2011)
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Fig. 2. Minimum inhibitory concentration of a mixture of heavy metals on single strains and bacterial mixtures (values are means ± SD).
during the induction of urea hydrolysis in bacterial mixtures of indigenous soil bacteria. During absorbance measurements, the stationary growth phase started 72 h after the start of the experiment (Fig. 4B), whereas during CFU measurements, the death phase started 48 h after the start of the experiment (Fig. 4C). In our experiment, the pH values increased rapidly for 48 h until they reached at an approximate pH value of 8.22 (Fig. 5A) because urea hydrolysis rates are higher in bacterial mixtures. However, during the CFU measurement, the death phase started 48 h after the start of the experiment (Fig. 5A). The removal of heavy metals by the bacterial mixtures was assessed using a 2 mM mixture of heavy metals, and the remediation potentials of the bacterial mixtures were observed to vary between 5.56% and 98.25% (Fig. 5B). Pb, Cd, and Cu concentrations were reduced to 0.06, 19.34, and 137.50 mg/L in about 48 h, respectively. This amounted to about 98.25%, 85.39%, and 5.56% Pb, Cd, and Cu removal by bioremediation, respectively. As previous studies have shown that
precipitated CaCO3 encapsulates bacteria cells (Mitchell and Ferris, 2006; Cuthbert et al., 2012), it is assumed that the electronegativity of the bacterial cell wall favors the adsorption of cations such as calcium ions, thus, facilitating the process of CaCO3 precipitation on the cell wall (Schultze-Lam et al., 1996). Various studies have also reported that CaCO3 may be the dominant sorbent for a variety of metals in carbonate aquifers (Zachara et al., 1991). Furthermore, heavy metals were reported to be sorbed on the surface of calcite (Lorens, 1981), with sorption being defined as a surface process irrespective of mechanism, adsorption, or precipitation (Sposito, 1984). 3.5. Column tests Bioremediation rates can be estimated based on the results from Fig. 6. The results showed that bioremediation rates increased in the following order: Cd (42.4%) < Cu (67.2%) < Pb (98.5%).
Fig. 3. Migration length of crystal violet under single strains and different bacterial mixtures (values are means ± SD). * Indicates P < 0.05.
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Fig. 5. Time dependency of growth pattern and heavy metal removal capacity of the bacterial mixtures.
Fig. 4. Changes over time in pH (A), OD at 600 nm (B), and colony forming unit (C) for treatments using different bacterial mixtures designs.
Adsorption affinity of heavy metals onto bacteria may be related to the electronegativity of metal ions as described by other studies (Merdy et al., 2009; Seco et al., 1997), which reported that attraction between metals and bacteria is higher with greater electronegativity. The selectivity in both single and multi-component solutions is higher for Pb2+ than for Cu2+ . In addition, the high ionic radius of ´˚ must induce a quick saturation of adsorption sites due Pb2+ (1.12 A) ´˚ These facts could to steric hindrance compared with Cu2+ (0.70 A). explain the reactivity of Cu and Pb with bacteria surface sites. Pb2+ adsorbs onto calcite and, thus, the Pb2+ ions move into Ca2+ sites, despite the large ionic radius of Pb2+ relative to Ca2+ (Sturchio et al., 1997). To further confirm the role of MICP in heavy metal bioremediation, remediated sand samples were analyzed by X-ray diffraction (XRD). XRD spectra indicated the presence of cerussite (lead carbonate, PbCO3 ), otavite (cadmium carbonate, CdCO3 ), and copper carbonate (CuCO3 ) crystals as the predominant minerals, as evidenced by the detection of sharp peaks in analysis of bioremediated samples, in contrast to the control, where peaks corresponding to quartz (SiO2 ) represented the sharpest peaks (Fig. 7).
Fig. 6. Removal of heavy metals by the bacterial mixtures (values are means ± SD).
The quartz peaks in the control samples might be attributed to the sand sample. A mixture of heavy metals was found to co-precipitate with CaCO3 as cerussite (lead carbonate, PbCO3 ) in the bioremediation experiments, while otavite (cadmium carbonate, CdCO3 ) and copper carbonate (CuCO3 ) peaks were also detected. The overall reactions involved in the bioremediation process include: production of NH4 + and HCO3 − by urease, desorption of Ca2+ and/or heavy metal ions from solid surfaces by the NH4 + and HCO3 − promoted precipitation of CaCO3 , and the co-precipitation of heavy metals.
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Fig. 7. XRD spectra, confirming the bioremediation of heavy metals by the bacterial mixtures grown in a sand column; XRD pattern of the control sand column (A) and bioremediated sand column (B). Identified peaks: C, CuCO3 ; O, otavite (CdCO3 ); P, cerussite (PbCO3 ); Q, quartz (SiO2 ).
4. Conclusion In this study, isolated strains were used to evaluate the synergistic effects of bacterial mixtures on bioremediation efficiency. At the end of the experimental period, the bacterial mixtures were found to be the most effective bacterial method, in comparison to individual culture methods. Compared with single strain cultures, the bacterial mixtures showed higher growth rate, urease activity, and resistance to heavy metals. Also, we demonstrated that the bacterial mixtures exhibited a considerably higher heavy metal bioremediation capacity than individual cultures, which might due to higher bacterial cell density at high levels of heavy metals. According to the column test results, the highest bioremediation efficiencies were noted for Pb, while the lowest removal efficiencies were detected for Cd. Bioremediation with the bacterial mixtures of isolated strains is an effective method for heavy metal removal from contaminated environments. This may prove to be a good strategy for developing an effective, efficient, and economic method of heavy metal bioremediation, and the introduction of these indigenous bacteria could be used for contaminated soil bioremediation without disturbing the target environment. 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. Akinci, G., Guven, D.E., 2011. Bioleaching of heavy metals contaminated sediment by pure and mixed cultures of Acidithiobacillus spp. Desalination 268, 221–226.
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