399
ARTICLE Biosorption of Cr(VI) ions from aqueous solutions by a newly isolated Bosea sp. strain Zer-1 from soil samples of a refuse processing plant Huining Zhang, Li Liu, Qing Chang, Hongyu Wang, and Kai Yang
Abstract: The adsorption behavior of Cr(VI) ions from aqueous solution by a chromium-tolerant strain was studied through batch experiments. An isolate designated Zer-1 was identified as a species of Bosea on the basis of 16S rRNA results. It showed a maximum resistance to 550 mg·L−1 Cr(VI). The effects of 3 important operating parameters, initial solution pH, initial Cr(VI) concentration, and biomass dose, were investigated by central composite design. On the basis of response surface methodology results, maximal removal efficiency of Cr(VI) was achieved under the following conditions: pH, 2.0; initial concentration of metal ions, 55 mg·L−1; and biomass dose, 2.0 g·L−1. Under the optimal conditions, the maximum removal efficiency of Cr(VI) ions was found to be nearly 98%. The experimental data exhibited a better fit with the Langmuir model than the Freundlich model. The biosorption mechanisms were investigated with pseudo-first-order, pseudo-second-order, and intraparticle diffusion kinetics models. These results revealed that biosorption of Cr(VI) onto bacterial biomass could be an alternative method for the removal of metal ions from aqueous solution. Key words: biosorption, Bosea sp., Cr(VI), isotherms, kinetics. Résumé : On a étudié le mode d’adsorption d’ions Cr(VI) en solution aqueuse par une souche tolérante au chrome en procédant a` des expériences en lots. On a identifié l’isolat Zer-1 comme une espèce de Bosea en vertu de sa séquence d’ARNr 16S; cet organisme faisait preuve d’une résistance maximale en présence de 550 mg·L–1 de Cr(VI). L’effet de 3 importants paramètres opérationnels, soit le pH initial de la solution, la concentration initiale de Cr(VI) et l’ajustement de la biomasse, a été examiné par planification expérimentale composite centrale. D’après les résultats du plan de surface de réponse, on a atteint l’efficacité maximale d’élimination du Cr(VI) dans les conditions suivantes : pH de 2,0; concentration initiale d’ions métalliques de 55 mg·L–1; et biomasse ajustée a` 2,0 g·L–1. Dans les conditions optimales, l’efficacité maximale d’élimination du Cr(VI) a frôlé les 98 %. On a noté que les données expérimentales s’adaptaient mieux a` un modèle de Langmuir qu’a` un modèle de Freundlich. On a étudié les mécanismes de biosorption en vertu de modèles de pseudo premier ordre, pseudo deuxième ordre et cinétique de diffusion intraparticulaire. Les résultats ont révélé que la biosorption du Cr(VI) sur la biomasse bactérienne pourrait représenter une solution de rechange pour éliminer les ions métalliques de solutions aqueuses. [Traduit par la Rédaction] Mots-clés : biosorption, Bosea sp., Cr(VI), isothermes, cinétiques.
Introduction Since the advent of industrialization and its rapid development, chromium (Cr) is widely utilized in many important industrial applications, such as leather tanning, chrome plating, pigmenting, wood preserving, textile dyeing, electroplating, and anodizing of aluminum (Zayed and Terry 2003). The wastewater resulting from these industrial activities contains high amounts of Cr, which has caused water and soil pollution as well as changes in the structures of natural communities and ecosystems. The toxicity, bioavailability, and mobility of Cr in environmental systems are highly dependent on its chemical species and oxidation states (Kimbrough et al. 1999; Zayed and Terry 2003). It is known that Cr occurs in the environment in several oxidation states, ranging from Cr(II) to Cr(VI), in which trivalent (Cr(III)) and hexavalent (Cr(VI)) are the 2 most common and stable chemical species. Cr(III) is considered to be a trace element essential for metabolic activities of living organisms. However, Cr(VI) is more water soluble than Cr(III) and is considered highly toxic, as it is also known to cause lung carcinoma and induce mutations and
cancer in humans and other biological communities (Kowalski 1994; Dixit et al. 2002; Gutierrez et al. 2012). Thus, the removal of Cr(VI) from wastewater before it is discharged into the soil–water system requires attention. The conventional methods for the removal of Cr(VI) ions from wastewater are usually achieved by chemical electrochemical treatment, electrolysis, ion exchange, precipitation, reverse osmosis, membrane separation, and evaporation (Han et al. 2007; Lameiras et al. 2008). However, in most cases, these processes might consume high amounts of energy, might be expensive, and might also be unfavorable and ineffective at lower metal ion concentration (Sudha Bai and Abraham 2001). Owing to the disadvantages of the conventional technologies, research focusing on alternative technologies and bioremediation of Cr(VI) has gained much attention since the first Cr(VI) reducing strain was isolated in the late 1970s. Biological treatment by using microorganisms is an alternative technique that possesses several advantages, including inexpensive, increased metal removal, regeneration of biosorbent, and also easy recovery of some valuable metals (Vijayaraghavan and Yun 2008). The use of microorganisms has become a popular method to remove Cr(VI) without
Received 28 October 2014. Revision received 21 March 2015. Accepted 23 March 2015. H. Zhang, L. Liu, Q. Chang, H. Wang, and K. Yang. School of Civil Engineering, Wuhan University, East Lake South Road 8, Wuchang District, Wuhan 430072, People’s Republic of China. Corresponding authors: Kai Yang (e-mail: [email protected]) and Hongyu Wang (e-mail: [email protected]). Can. J. Microbiol. 61: 399–408 (2015) dx.doi.org/10.1139/cjm-2014-0719
Published at www.nrcresearchpress.com/cjm on 25 March 2015.
400
generating harmful secondary pollution. During the past 3 decades, many studies have discussed a wide variety of microorganisms, such as algae, fungi, protozoa, yeast, and bacteria, that are widely used for sorptive removal of metal ions. Among the microorganisms, bacteria are better candidates, since they can be easily cultured with simple nutrients and are easy to handle (Oh et al. 2011). Numerous bacteria, such as Aeromonas spp., Bacillus spp., Pseudomonas spp., Staphylococcus spp., and Zooglea spp., have been reported to adsorb Cr(VI) from aqueous solution (Dixit et al. 2002; Loukidou et al. 2004; Zhou et al. 2007; Ziagova et al. 2007). However, to the best of our knowledge, the adsorption capacity of Bosea sp. has seldom been reported. In the current study, the Cr(VI)-resistant pure strain Bosea sp. strain Zer-1 was isolated, and its adsorption of Cr(VI) ions from aqueous solutions was studied through batch experiments. Three important operating parameters, i.e., the initial solution pH, initial Cr(VI) concentration, and biomass dose, affecting the adsorption process were investigated by central composite design (CCD). The optimal conditions for Cr(VI) removal were established by using response surface methodology (RSM). Equilibrium isotherm and kinetics models were constructed to explore the biosorption mechanism.
Materials and methods Bacterial isolation and culture medium Soil samples were collected from Chen Guchong refuse processing plant (Wuhan, China). The samples were diluted in sterile 0.9% NaCl solution and then streaked onto nutrient medium plates with 15 g·L−1 agar and 25 mg·L−1 Cr(VI). Inoculated plates were incubated for 48 h at 30 °C and observed for bacterial growth. Colonies surviving on the plate were evaluated as Cr(VI)-resistant strains. The morphologically distinct colonies were selected and purified by repeated streaking on agar plates with Cr(VI). Nutrient medium contained peptone, 10 g·L−1; beef extract, 5 g·L−1; NaCl, 5 g·L−1; distilled water, 1 L; pH 7.0 ± 0.2. The metal ion Cr(VI) was used in the form of K2Cr2O7. Minimal inhibitory concentration of Cr(VI) The minimal inhibitory concentration (MIC) of Cr(VI) was determined according to Hassen et al. (1998). Experiments were carried out in 500 mL Erlenmeyer flasks containing 20 mL of pure culture of isolates and 200 mL of nutrient medium in which increasing concentrations of Cr(VI) (25–1000 mg·L−1) were supplemented. The flasks were incubated at 30 °C for 48 h and shaken at 120 r·min–1 (1.6g). The MIC is expressed as the minimum concentration of Cr(VI) that completely inhibited the growth of bacteria. Bacterial growth was monitored from the optical density values at 600 nm (OD600) using a spectrophotometer (Nanbeijt, China). The isolate that had the maximum tolerance to Cr(VI) was designated as Zer-1 and chosen for further studies. Identification of isolate Zer-1 To identify the isolate Zer-1, physiological and biochemical characteristics were tested by Sangon Biotech Co., Ltd. (Shanghai, China). Cell morphology was observed by scanning electron microscopy. Further identification was performed via 16S rRNA gene sequencing. Genomic DNA was extracted using a DNA rapid extraction kit (Sangon, Shanghai, China) according to the manufacturer’s instructions. The 16S rRNA gene was amplified by PCR using universal bacterial primers (338F, 5=-ACTCCTACGGGAGGCAGCAG-3=; 534R, 5=-ATTACCGCGGCTGCTGG-3=). The PCR mixture contained 1 L of DNA template, 1 L of each primer, 1 L of deoxyribonucleotide triphosphate (dNTP) (2.5 mmol·L–1), 10 L of 10× PCR buffer, 0.25 L of rTaq DNA Polymerase (5 U·L−1), and 35.75 L of sterilized deionized water was added. The settings for PCR were as follows: initial denaturation for 2 min at 95 °C, followed by 30 cycles of denaturation and annealing (30 s at 95 °C,
Can. J. Microbiol. Vol. 61, 2015
Table 1. Experimental design based on the central composite design.
No.
Factor A — pH
Factor B — initial Cr(VI) concn. (mg·L–1)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
4.5 7.0 8.7 7.0 2.0 4.5 2.0 4.5 4.5 4.5 2.0 4.5 2.0 7.0 4.5 4.5 7.0 4.5 2.0 4.5
50.00 50.00 187.50 325.00 50.00 418.70 187.50 187.50 187.50 187.50 50.00 187.50 325.00 50.00 187.50 187.50 325.00 187.50 325.00 187.50
Factor C — adsorbent dosage (g·L–1) 1.60 0.20 1.60 3.00 0.20 1.60 1.60 0.20 1.60 1.60 3.00 1.60 0.20 3.00 3.90 1.60 0.20 1.60 3.00 1.60
Biosorption efficiency (%) Actual value
Predicted value
67.30 47.30 40.50 55.30 57.10 48.20 97.30 12.60 66.00 65.30 99.30 66.40 9.80 53.50 67.70 67.10 6.60 64.90 96.80 66.80
74.82 40.64 47.02 55.18 56.00 47.27 83.74 25.12 65.33 65.33 103.04 65.34 11.50 50.53 65.00 65.34 1.64 65.34 102.23 65.34
30 s at 58 °C, 180 s at 72 °C), and a final extension at 72 °C for 10 min. The amplified PCR product was purified by Agarose Gel DNA Purification kit (Watson, Shanghai, China). To determine the closest matches, the sequences were searched in the GenBank database using BLAST program. The phylogenetic tree was constructed by using the neighbor-joining algorithm in the MEGA 6.06 program. Preparation of biosorbent The isolate Zer-1 was enriched in nutrient medium for 48 h at 30 °C and shaken at 120 r·min–1. After incubation, the cells were harvested by centrifugation at 8000 r·min–1 (7155g) for 10 min and washed 3 times with deionized water, then used as biosorbent. Optimization of Cr(VI) adsorption To determine the optimal conditions for the adsorption of Cr(VI), 3 important operating variables were selected for RSM: initial solution pH (2.0–7.0), initial Cr(VI) concentration (50– 300 mg·L−1), and biosorbent dose (0.2–3.0 g dry mass·L−1). The optimization of the removal efficiency of Cr(VI) was performed by CCD with 20 groups of independent experiments (Table 1). For the statistical calculations, the relationship between the real values and the coded ones was calculated by Design Expert 8.0. Each coefficient was analyzed by analysis of variance (ANOVA). If the P value (Pr >F) was F)
Model A B C AB AC BC A2 B2 C2
12322.13 2571.27 1046.06 6677.29 14.85 690.06 952.66 68.65 1.92 1983.53
9 1 1 1 1 1 1 1 1 1
1369.13 2571.27 1046.06 6677.29 14.85 690.06 952.66 68.65 1.92 1983.53
23.59 44.30 18.02 115.05 0.26 11.89 16.41 1.18 0.03 34.18
ARTICLE Biosorption of Cr(VI) ions from aqueous solutions by a newly isolated Bosea sp. strain Zer-1 from soil samples of a refuse processing plant Huining Zhang, Li Liu, Qing Chang, Hongyu Wang, and Kai Yang
Abstract: The adsorption behavior of Cr(VI) ions from aqueous solution by a chromium-tolerant strain was studied through batch experiments. An isolate designated Zer-1 was identified as a species of Bosea on the basis of 16S rRNA results. It showed a maximum resistance to 550 mg·L−1 Cr(VI). The effects of 3 important operating parameters, initial solution pH, initial Cr(VI) concentration, and biomass dose, were investigated by central composite design. On the basis of response surface methodology results, maximal removal efficiency of Cr(VI) was achieved under the following conditions: pH, 2.0; initial concentration of metal ions, 55 mg·L−1; and biomass dose, 2.0 g·L−1. Under the optimal conditions, the maximum removal efficiency of Cr(VI) ions was found to be nearly 98%. The experimental data exhibited a better fit with the Langmuir model than the Freundlich model. The biosorption mechanisms were investigated with pseudo-first-order, pseudo-second-order, and intraparticle diffusion kinetics models. These results revealed that biosorption of Cr(VI) onto bacterial biomass could be an alternative method for the removal of metal ions from aqueous solution. Key words: biosorption, Bosea sp., Cr(VI), isotherms, kinetics. Résumé : On a étudié le mode d’adsorption d’ions Cr(VI) en solution aqueuse par une souche tolérante au chrome en procédant a` des expériences en lots. On a identifié l’isolat Zer-1 comme une espèce de Bosea en vertu de sa séquence d’ARNr 16S; cet organisme faisait preuve d’une résistance maximale en présence de 550 mg·L–1 de Cr(VI). L’effet de 3 importants paramètres opérationnels, soit le pH initial de la solution, la concentration initiale de Cr(VI) et l’ajustement de la biomasse, a été examiné par planification expérimentale composite centrale. D’après les résultats du plan de surface de réponse, on a atteint l’efficacité maximale d’élimination du Cr(VI) dans les conditions suivantes : pH de 2,0; concentration initiale d’ions métalliques de 55 mg·L–1; et biomasse ajustée a` 2,0 g·L–1. Dans les conditions optimales, l’efficacité maximale d’élimination du Cr(VI) a frôlé les 98 %. On a noté que les données expérimentales s’adaptaient mieux a` un modèle de Langmuir qu’a` un modèle de Freundlich. On a étudié les mécanismes de biosorption en vertu de modèles de pseudo premier ordre, pseudo deuxième ordre et cinétique de diffusion intraparticulaire. Les résultats ont révélé que la biosorption du Cr(VI) sur la biomasse bactérienne pourrait représenter une solution de rechange pour éliminer les ions métalliques de solutions aqueuses. [Traduit par la Rédaction] Mots-clés : biosorption, Bosea sp., Cr(VI), isothermes, cinétiques.
Introduction Since the advent of industrialization and its rapid development, chromium (Cr) is widely utilized in many important industrial applications, such as leather tanning, chrome plating, pigmenting, wood preserving, textile dyeing, electroplating, and anodizing of aluminum (Zayed and Terry 2003). The wastewater resulting from these industrial activities contains high amounts of Cr, which has caused water and soil pollution as well as changes in the structures of natural communities and ecosystems. The toxicity, bioavailability, and mobility of Cr in environmental systems are highly dependent on its chemical species and oxidation states (Kimbrough et al. 1999; Zayed and Terry 2003). It is known that Cr occurs in the environment in several oxidation states, ranging from Cr(II) to Cr(VI), in which trivalent (Cr(III)) and hexavalent (Cr(VI)) are the 2 most common and stable chemical species. Cr(III) is considered to be a trace element essential for metabolic activities of living organisms. However, Cr(VI) is more water soluble than Cr(III) and is considered highly toxic, as it is also known to cause lung carcinoma and induce mutations and
cancer in humans and other biological communities (Kowalski 1994; Dixit et al. 2002; Gutierrez et al. 2012). Thus, the removal of Cr(VI) from wastewater before it is discharged into the soil–water system requires attention. The conventional methods for the removal of Cr(VI) ions from wastewater are usually achieved by chemical electrochemical treatment, electrolysis, ion exchange, precipitation, reverse osmosis, membrane separation, and evaporation (Han et al. 2007; Lameiras et al. 2008). However, in most cases, these processes might consume high amounts of energy, might be expensive, and might also be unfavorable and ineffective at lower metal ion concentration (Sudha Bai and Abraham 2001). Owing to the disadvantages of the conventional technologies, research focusing on alternative technologies and bioremediation of Cr(VI) has gained much attention since the first Cr(VI) reducing strain was isolated in the late 1970s. Biological treatment by using microorganisms is an alternative technique that possesses several advantages, including inexpensive, increased metal removal, regeneration of biosorbent, and also easy recovery of some valuable metals (Vijayaraghavan and Yun 2008). The use of microorganisms has become a popular method to remove Cr(VI) without
Received 28 October 2014. Revision received 21 March 2015. Accepted 23 March 2015. H. Zhang, L. Liu, Q. Chang, H. Wang, and K. Yang. School of Civil Engineering, Wuhan University, East Lake South Road 8, Wuchang District, Wuhan 430072, People’s Republic of China. Corresponding authors: Kai Yang (e-mail: [email protected]) and Hongyu Wang (e-mail: [email protected]). Can. J. Microbiol. 61: 399–408 (2015) dx.doi.org/10.1139/cjm-2014-0719
Published at www.nrcresearchpress.com/cjm on 25 March 2015.
400
generating harmful secondary pollution. During the past 3 decades, many studies have discussed a wide variety of microorganisms, such as algae, fungi, protozoa, yeast, and bacteria, that are widely used for sorptive removal of metal ions. Among the microorganisms, bacteria are better candidates, since they can be easily cultured with simple nutrients and are easy to handle (Oh et al. 2011). Numerous bacteria, such as Aeromonas spp., Bacillus spp., Pseudomonas spp., Staphylococcus spp., and Zooglea spp., have been reported to adsorb Cr(VI) from aqueous solution (Dixit et al. 2002; Loukidou et al. 2004; Zhou et al. 2007; Ziagova et al. 2007). However, to the best of our knowledge, the adsorption capacity of Bosea sp. has seldom been reported. In the current study, the Cr(VI)-resistant pure strain Bosea sp. strain Zer-1 was isolated, and its adsorption of Cr(VI) ions from aqueous solutions was studied through batch experiments. Three important operating parameters, i.e., the initial solution pH, initial Cr(VI) concentration, and biomass dose, affecting the adsorption process were investigated by central composite design (CCD). The optimal conditions for Cr(VI) removal were established by using response surface methodology (RSM). Equilibrium isotherm and kinetics models were constructed to explore the biosorption mechanism.
Materials and methods Bacterial isolation and culture medium Soil samples were collected from Chen Guchong refuse processing plant (Wuhan, China). The samples were diluted in sterile 0.9% NaCl solution and then streaked onto nutrient medium plates with 15 g·L−1 agar and 25 mg·L−1 Cr(VI). Inoculated plates were incubated for 48 h at 30 °C and observed for bacterial growth. Colonies surviving on the plate were evaluated as Cr(VI)-resistant strains. The morphologically distinct colonies were selected and purified by repeated streaking on agar plates with Cr(VI). Nutrient medium contained peptone, 10 g·L−1; beef extract, 5 g·L−1; NaCl, 5 g·L−1; distilled water, 1 L; pH 7.0 ± 0.2. The metal ion Cr(VI) was used in the form of K2Cr2O7. Minimal inhibitory concentration of Cr(VI) The minimal inhibitory concentration (MIC) of Cr(VI) was determined according to Hassen et al. (1998). Experiments were carried out in 500 mL Erlenmeyer flasks containing 20 mL of pure culture of isolates and 200 mL of nutrient medium in which increasing concentrations of Cr(VI) (25–1000 mg·L−1) were supplemented. The flasks were incubated at 30 °C for 48 h and shaken at 120 r·min–1 (1.6g). The MIC is expressed as the minimum concentration of Cr(VI) that completely inhibited the growth of bacteria. Bacterial growth was monitored from the optical density values at 600 nm (OD600) using a spectrophotometer (Nanbeijt, China). The isolate that had the maximum tolerance to Cr(VI) was designated as Zer-1 and chosen for further studies. Identification of isolate Zer-1 To identify the isolate Zer-1, physiological and biochemical characteristics were tested by Sangon Biotech Co., Ltd. (Shanghai, China). Cell morphology was observed by scanning electron microscopy. Further identification was performed via 16S rRNA gene sequencing. Genomic DNA was extracted using a DNA rapid extraction kit (Sangon, Shanghai, China) according to the manufacturer’s instructions. The 16S rRNA gene was amplified by PCR using universal bacterial primers (338F, 5=-ACTCCTACGGGAGGCAGCAG-3=; 534R, 5=-ATTACCGCGGCTGCTGG-3=). The PCR mixture contained 1 L of DNA template, 1 L of each primer, 1 L of deoxyribonucleotide triphosphate (dNTP) (2.5 mmol·L–1), 10 L of 10× PCR buffer, 0.25 L of rTaq DNA Polymerase (5 U·L−1), and 35.75 L of sterilized deionized water was added. The settings for PCR were as follows: initial denaturation for 2 min at 95 °C, followed by 30 cycles of denaturation and annealing (30 s at 95 °C,
Can. J. Microbiol. Vol. 61, 2015
Table 1. Experimental design based on the central composite design.
No.
Factor A — pH
Factor B — initial Cr(VI) concn. (mg·L–1)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
4.5 7.0 8.7 7.0 2.0 4.5 2.0 4.5 4.5 4.5 2.0 4.5 2.0 7.0 4.5 4.5 7.0 4.5 2.0 4.5
50.00 50.00 187.50 325.00 50.00 418.70 187.50 187.50 187.50 187.50 50.00 187.50 325.00 50.00 187.50 187.50 325.00 187.50 325.00 187.50
Factor C — adsorbent dosage (g·L–1) 1.60 0.20 1.60 3.00 0.20 1.60 1.60 0.20 1.60 1.60 3.00 1.60 0.20 3.00 3.90 1.60 0.20 1.60 3.00 1.60
Biosorption efficiency (%) Actual value
Predicted value
67.30 47.30 40.50 55.30 57.10 48.20 97.30 12.60 66.00 65.30 99.30 66.40 9.80 53.50 67.70 67.10 6.60 64.90 96.80 66.80
74.82 40.64 47.02 55.18 56.00 47.27 83.74 25.12 65.33 65.33 103.04 65.34 11.50 50.53 65.00 65.34 1.64 65.34 102.23 65.34
30 s at 58 °C, 180 s at 72 °C), and a final extension at 72 °C for 10 min. The amplified PCR product was purified by Agarose Gel DNA Purification kit (Watson, Shanghai, China). To determine the closest matches, the sequences were searched in the GenBank database using BLAST program. The phylogenetic tree was constructed by using the neighbor-joining algorithm in the MEGA 6.06 program. Preparation of biosorbent The isolate Zer-1 was enriched in nutrient medium for 48 h at 30 °C and shaken at 120 r·min–1. After incubation, the cells were harvested by centrifugation at 8000 r·min–1 (7155g) for 10 min and washed 3 times with deionized water, then used as biosorbent. Optimization of Cr(VI) adsorption To determine the optimal conditions for the adsorption of Cr(VI), 3 important operating variables were selected for RSM: initial solution pH (2.0–7.0), initial Cr(VI) concentration (50– 300 mg·L−1), and biosorbent dose (0.2–3.0 g dry mass·L−1). The optimization of the removal efficiency of Cr(VI) was performed by CCD with 20 groups of independent experiments (Table 1). For the statistical calculations, the relationship between the real values and the coded ones was calculated by Design Expert 8.0. Each coefficient was analyzed by analysis of variance (ANOVA). If the P value (Pr >F) was F)
Model A B C AB AC BC A2 B2 C2
12322.13 2571.27 1046.06 6677.29 14.85 690.06 952.66 68.65 1.92 1983.53
9 1 1 1 1 1 1 1 1 1
1369.13 2571.27 1046.06 6677.29 14.85 690.06 952.66 68.65 1.92 1983.53
23.59 44.30 18.02 115.05 0.26 11.89 16.41 1.18 0.03 34.18
