- Email: [email protected]
Bioreduction of chromium (VI) by Bacillus sp. isolated from soils of iron mineral area
European Journal of Soil Biology 45 (2009) 483–487
Contents lists available at ScienceDirect
European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi
Original article
Bioreduction of chromium (VI) by Bacillus sp. isolated from soils of iron mineral area Guojun Cheng*, Xiaohua Li Key Lab for Microorganism and Biological Transformation, College of Life Science, South-Central University for Nationalities, 430074 Wuhan, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 June 2008 Received in revised form 24 January 2009 Accepted 19 June 2009 Available online 7 August 2009 Handling editor: Hermann Verhoef
Extensive use of chromium in industry has caused environmental contamination. Chromium-resistant bacteria are capable of reducing toxic Cr (VI) to less toxic Cr (III). Eight isolates, which can grow on LB agar containing 500 mg/L of Cr (VI), were isolated from soil samples of iron mineral area. The bacterial isolates were identified as Bacillus sp. by the 16S rRNA gene sequences. Phylogenetic tree analysis indicates the isolates can be divided into two groups. The bacterial isolates can be resistant to other heavy metals and reduce Cr (VI) at different levels. One bacterial isolate (MDS05), which can tolerate 2500 mg/L Cr (VI) and was able to reduce almost 100% of Cr (VI) at the concentration of 10 mg/L in 24 h, was selected to study the effects of some environmental factors such as pH, temperature, and time on Cr (VI) reduction and growth. The cell growth of MDS05 was affected by the presence of Cr (VI), especially at the concentration of 100 mg/ L. It reduced more amount of Cr (VI) under a wide range of concentrations from 5 to 50 mg/L, and reduction was optimum at 37 C and pH 8. MDS05 showed great promise for use in Cr (VI) detoxification under a wide range of environmental conditions. Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords: Bacillus sp. Cr (VI) reduction Metal resistance Bioremediation
1. Introduction Chromate has many industrial applications and defense applications such as stainless steel, chrome plating, leather tanning, dyes, pigments and nuclear weapons production, and often causes a widespread environmental contaminant [1]. It generally exists in two stable oxidation states, trivalent or chromium III and hexavalent or chromium VI. The trivalent chromium is less toxic and mobile, while the hexavalent form of chromium, Cr (VI), is highly soluble and a strong oxidizing agent that is reduced intracellularly to Cr5þ and reacts with nucleic acids and other cell components to produce mutagenic and carcinogenic effects on biological systems [2]. Hence, reduction of Cr (VI) to Cr (III) is an attractive and useful method for Cr (VI) pollution [3]. Conventional physicochemical methods for removing toxic CrO24 include electrochemical treatment, precipitation, ion exchange, evaporation, reverse osmosis, and adsorption on activated coal. However, most of these methods are often inefficient and very expensive especially for metals at low concentration [2,4], and hence biological approach has been considered as an alternative remediation for chromium(VI) contamination due to the lower costs and the significant smaller quantities of the produced sludge [5].
* Corresponding author. Fax: þ86 27 67842689. E-mail address: [email protected] (G. Cheng). 1164-5563/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejsobi.2009.06.009
Biological approach could be initiated by the microbial reduction of Cr (VI). Microbial populations in Cr (VI) polluted environments adapt to toxic concentrations of Cr (VI) and become chromium (VI)-resistant and chromium (VI)-reducing strains [6]. In previous researches, a number of microorganisms have been reported to be able to reduce Cr (VI), including strains of Pseudomonas [7,8], Bacillus [9], Enterobacter [10,11], Escherichia coli [12], Shewanella [13,14], and several other bacterial isolates [15,16]. The objectives of this study was to isolate bacteria from iron mineral area where the hexavalent chromium and other heavy metal level is quite high, to measure the bacterial minimum inhibitory concentrations (MIC) of heavy metals, and to research the characterization of factors involved in hexavalent chromium reduction. These results obtained in this study may provide the useful knowledge for the bioremediation of chromate pollution. 2. Materials and methods 2.1. Soil samples and isolation of bacteria Soil samples were collected from iron mineral area in HuBei Province, China that proved many minerals such as iron, manganese, chromium and copper. For the isolation of bacteria, soil samples were serially diluted and plated onto Luria–Bertani (LB) agar. The molten medium was supplemented with Cr (VI) as K2CrO4 to final concentration 500 mg/L [2]. The Cr(VI) stock solution was
484
G. Cheng, X. Li / European Journal of Soil Biology 45 (2009) 483–487
filter sterilized with a 0.22 mm membrane filter. Plates were incubated at 37 C for 48 h. Colonies of different morphologies were then selected on LB agar containing 500 mg/L of Cr (VI) and incubated at 37 C for 24 h. 2.2. Identification of chromium-resistant bacteria The bacterial isolates were identified by 16S rRNA gene as follows. Bacterial strains were grown in LB broth supplemented with 500 mg/L of Cr (VI) at 37 C for 48 h and DNA was extracted and purified according to the method of Asubel et al. [7]. The 16S rRNA gene was PCR amplified from the isolated DNA with the following primers of 16S-L (5’AAGGAGGTGATCCAGCC3’) and 16S-R (5’AGAGTTTGATCCTGGCT CAG3’). The thermocycling conditions were as follows: 3 min at 94 C; followed by 30 cycles of 1 min at 94 C, 1 min at 56 C, and 2 min at 72 C; followed by 10 min at 72 C and a hold at 4 C. PCR products were obtained and cloned into plasmid vector pMD-18T (Tarkara, DaLian, China) and then sequenced. The nucleotides of 16S rRNA gene were carried out using BLAST (http://www.ncbi.nlm.nih.gov/BLAST). Phylogenetic tree of small Chromium-Resistant Bacteria based on 16S rRNA genes was constructed using the minimum-evolution method of Mega 4.0 software [17]. 2.3. Determination of minimum inhibitory concentrations (MIC) of heavy metals The medium used for MIC measurement was LB agar medium containing heavy metal salts in varying concentrations ranging from 50 to 3000 mg/L. The plates were checked for growth after 3 d incubation at 37 C [18]. The MIC was defined as the lowest metal concentration that completely inhibited growth. The following metal salts were used: K2CrO4, CdCl2$2.5H2O, ZnSO4$7H2O, NiSO4, CuSO4$5H2O, MnCl2$4H2O, and Pb(C2H3O2)$3H2O. 2.4. Characterization of chromium reduction by isolates Chromate-resistant bacterial isolates were inoculated into LB broth (pH 7.0) containing 10 mg/L of Cr (VI) and incubated for 24 h at 37 C with orbital shaking at 200 rpm [3]. Bacterial cell density of the liquid cultures was routinely monitored by measuring optical density (OD) at 600 nm. Bioreduction of Cr (VI) was done according to the procedure of Hernandez et al. [19]. Hexavalent chromium was determined spectrophotometrically at a wavelength of 540 nm with the 1,5-diphenylcarbazide (DPC) method [20]. 2.5. Effect of pH and temperature on chromate reduction The influence of temperature and pH on bacterial chromate reduction was assessed. For the effect of pH, the pH value of the autoclaved culture medium was adjusted to 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 with predetermined amounts of filter-sterilized 10% (w/v) NaOH and 10% (w/v) HCl at 37 C. Incubation temperature was varied at 20, 30, 37, 40 and 45 C. Bacterial isolates were inoculated at an initial optical density at 600 nm (OD600) of 0.08–0.17 into fresh LB medium containing 10 mg/L of Cr (VI).
Table 1 Identification of chromium-resistant bacteria using 16S rRNA gene sequence. Isolates
Accession no.
Similarity
Species as close relatives
MDS01 MDS02 MDS03 MDS04 MDS05 MDS06 MDS07 MDS08
EU236670 EU236671 EU236672 EU236674 EU236673 EU236675 EU236676 EU236677
99% 96% 97% 97% 99% 97% 97% 98%
Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus
indicus badius megaterium flexus megaterium thuringiensis megaterium luciferensis
PCR amplification of the 16S rRNA genes, which were then sequenced, and the nucleotide sequences were analyzed for homology using BlastN (Table 1). The results indicated that the eight 16S rRNA genes all showed more than 96% sequence homology to those in the genus Bacillus. This may imply that Bacillus sp. can tolerate the toxicity of Cr (VI) and probably have become the dominant strains in the high level of heavy metal contamination area. 3.2. Construction of phylogenetic tree A phylogenetic tree was constructed based on 16S rRNA partial sequences (Fig. 1). The results showed that the 8 isolates can be divided into two groups, the isolates MDS01, MDS06, MDS08, stood well apart from strains MDS02, MDS03, MDS04,. MDS05, MDS07. The difference in species grouping may suggest a differential rate of evolutionary divergence among the chromate-resistant Bacillus species. 3.3. Determination of heavy metal resistance All the bacterial isolates were tested for their resistance against different metal ions that are Cr6þ, Cd2þ, Zn2þ, Ni2þ, Cu2þ, Pb2þ and Mn2þ. As shown in Table 2, all isolated strains showed no growth inhibition up to 500 mg/L Cr (VI), and MDS05 even could grow with 2500 mg/L Cr (VI). The bacterial isolates also demonstrated an attractive resistance to other heavy metals (Table 2). The MIC values ranged from about 50 mg/L (Cu2þ) to 2500 mg/L (Cr6þ). From comparisons of the results across the metals, MDS05 and MDS02 exhibited a biggish MIC value of the heavy metals, and their resistance levels were considerably greater than those reported for other bacteria [19,21]. A high degree of tolerance to multiple heavy metals is necessary for the capacity of the strains to survive in different sources of pollution with elevated heavy metal levels [22]. Chromate reduction activity of whole cells was detected in all isolates at initial Cr (VI) concentration of 10 mg/L (Fig. 2). The ability of MDS02 and MDS05 to reduce Cr (VI) in 24 h period is of interest as it is relatively fast compared to other species. MDS02 and MDS05 showed a reduction of 59% and 65%, respectively, after 6 h of
3. Results 3.1. Screening and identification of bacterial isolates Eight isolates (MDS01-MDS08) with inherent ability of 500 mg/L Chromate resistance have been isolated from the soils of iron mineral area in HuBei Province, China. The isolates showed significant growth in LB medium containing 500 mg/L Cr (VI). All the isolates formed spores and were gram-positive. The isolates were identified by initial
Fig. 1. Phylogenetic tree derived from 16S rRNA gene sequences of chromium-resistant bacteria. The scale bar corresponds to 0.005 substitution per nucleotide position.
G. Cheng, X. Li / European Journal of Soil Biology 45 (2009) 483–487
485
Table 2 Resistance to heavy metals in chromium-resistant bacterial isolates. Stains
MICs (mg/L) for Bacillus. sp strains Cr6þ
Cd2þ
Zn2þ
Ni2þ
Cu2þ
Mn2þ
Pb2þ
MDS01 MDS02 MDS03 MDS04 MDS05 MDS06 MDS07 MDS08
1000 2000 1500 500 2500 500 500 1000
200 300 200 100 400 200 100 200
400 800 300 600 500 400 500 200
200 300 500 300 700 200 200 100
100 100 200 50 200 300 100 100
400 500 300 400 600 300 200 300
1000 2000 500 1500 2000 500 100 500
incubation at Cr (VI) concentration of 10 mg/L. On the next 6 h, 21% and 18%, respectively, of the added Cr (VI) was reduced, and after 24 h, more than 99% of Cr (VI) was reduced. Isolate MDS05 was selected for further studies due to the relatively higher Cr (VI) reduction and higher resistance levels of heavy metals in this strain. 3.4. Effect of Cr (VI) on cell growth The effect of Cr (VI) on the growth of MDS05 was evaluated before the experiments of Cr (VI) reduction. Fig. 3 shows MDS05 was exposed to six different Cr (VI) concentrations, and bacterial density (OD600) was measured after 24 h. It is obvious that Cr (VI) at 1, 5, 10 and 50 mg/l has slight effect on the growth of cells. However, growth was inhibited by 100 mg/L of Cr (VI) and decreased cell density by about 20%. The growth curves of MDS05 in LB medium with or without 10 mg/ l Cr (VI) is shown in Fig. 4. The growth curve of MDS05 in the medium containing Cr (VI) did not follow the same growth pattern as the control. Growth of MDS05 in the presence of 10 mg/l Cr (VI) showed a longer lag phase than that in the absence of Cr (VI). It indicates that the toxic Cr (VI) effect cell growth during the incubation time of 24 h. 3.5. Factors affecting on chromium reduction Initial pH of the medium with 10 mg/L Cr (VI) supplied was considered as a factor for Cr (VI) reduction by MDS05 (Fig. 5). Values for pH of 5 and 10 restricted bacterial Cr (VI) reduction, while at pH ranging from 7 to 9, MDS05 had high activity for Cr (VI) reduction. The optimum initial pH value was 8. As at pH 8, almost complete Cr (VI) was reduced during the incubation time of 24 h. Microbial Cr (VI) reduction was dependent on temperature. Cr (VI) reduction by MDS05 strain was investigated over four different temperatures: 20, 30, 37 and 45 C (Fig. 6). Cr (VI) was reduced effectively at 30 and 37 C, with reduction 91% and 100%, respectively.
Fig. 2. Cr (VI) reduction of Cr (VI)-resistant bacteria. The cells were cultured on Luria– Bertani agar supplemented with 10 mg/L Cr (VI). The Cr (VI) reduction activity was measured after incubation for 0, 6, 12, 18, 24 h at 37 C. Error bars represent standard deviation (n 1).
Fig. 3. Growth of Bacillus sp. MDS05. The cells were cultured on Luria–Bertani agar supplemented with 0, 1, 5, 10, 50, 100 and 300 mg/L Cr (VI), respectively. The optical density was measured after incubation for 24 h at 37 C. Error bars represent standard deviation (n 1).
While at 20 and 45 C, chromate reduction was severely decreased. This indicates that MDS05 strain reduced Cr (VI) better at 37 C. The Cr (VI)-resistant bacterial MDS05 was exposed to six different Cr (VI) concentrations, and Cr (VI) reduction was evaluated (Fig. 7). At initial concentration of 5, 10 and 50 mg/l, MDS05 strain showed the highest Cr (VI) reduction rate, while a sharp decrease was observed in presence of a high initial concentration of Cr (VI) at 100 mg/L. 4. Discussion Metal-polluted environments pose serious health and ecological risk. Metal containing industrial effluents constitute a major source of metallic pollution [23]. Soil is a common environment for Cr(VI) contamination, and most Cr (VI)-resistant bacteria reported to date have been isolated from soil. Here we describe the identification of 8 Cr(VI)-resistant bacterial isolates and the characterization of Cr(VI) reduction by selected isolate. Most of the isolates were able to reduce Cr(VI), but at different rates. The isolates all belonged to the genera Bacillus. Few isolates of the genus Bacillus have been known to tolerate and reduce Cr(VI) [9,24,25]. The tolerance level of Cr(VI) for the new Bacillus sp. MDS05 was 2500 mg/L, which is more than other strain of Bacillus sp. those have been known to tolerate no more than 2500 mg/L Cr(VI). However, a bacterial strain (probably a Bacillus species) from a tannery effluent has been reported which is resistant to very high level of Cr(VI) at more than 8 mg/mL [26].
Fig. 4. Growth curves of Bacillus sp. MDS05 in the absence or in the presence of 10 mg/L Cr (VI). Error bars represent standard deviation (n 1).
486
G. Cheng, X. Li / European Journal of Soil Biology 45 (2009) 483–487
Fig. 5. Effect of pH on Cr (VI) reduction of Bacillus sp. MDS05. Initial pH of the culture medium was adjusted to 5–10. The Cr (VI) reduction activity was measured after incubation for 24 h at 37 C. Error bars represent standard deviation (n 1).
Fig. 7. Effect of initial Cr (VI) concentration on Cr (VI) reduction of Bacillus sp. MDS05. The Cr (VI) reduction activity was measured after incubation for 24 h at pH 8 and 37 C. Error bars represent standard deviation (n 1).
The strain MDS05 was shown to be of a high efficiency in detoxifying chromate. It could rapidly reduce almost 100% of 10 mg/ L Cr(VI) with 24 h. Pal and Paul [27] reported an isolate B. sphaericus AND303 has high Cr(VI) removal efficiency, and it can removed about 72% Cr(VI) (10 mg/L) from the medium within 24 h. Liu et al. [3] also reported that Bacillus sp. XW-4 was capable of removing 100% of 10 mg/L in 24 h, but it can only tolerate 100 mg/L Cr (VI). Cr(VI) reduction were dependent on pH, temperature, and Cr concentration. The optimum pH for growth of the isolate MDS05 was 7.0–9.0. Extreme pH (5.0, 6.0 and 10.0) restricted Cr(VI) reduction. Variation of the culture initial pH highly affected Cr(VI) reduction. Camargo et al. [2] reported that optimal pH for Cr (VI) reduction by five chromate bacteria was 7–8. Wang and Xiao [28] also reported that optimal pH for Cr (VI) reduction of Bacillus sp. and Pseudomonas fluorescence was 7, but it was strongly inhibited at pH 6. The change in optimal pH indicates that pH modification is important for different cultures to achieve the maximum Cr(VI) reduction in Cr (VI) detoxification. Temperature is also an important factor for bacterial Cr(VI) reduction. The optimum of the tested temperatures was 37 C for Cr(VI) reduction of MDS05. Extreme temperature (20 and 45 C) restricted Cr(VI) reduction. Optimal temperature for Cr (VI) reduction of Cr (VI)-resistant bacteria was reported at 37 C, but it was almost ceased at 20 C [29]. Wang et al. [30] reported that no chromate reduction was observed at 4 and 60 C. Since Cr (VI) reduction is enzyme-mediated, changes in temperature will affect the enzyme activity. In addition, the results clearly showed a direct positive correlation between Cr(VI) reduction and bacterial growth with Cr(VI) concentration of 1–100 mg/L. Both growth and Cr (VI) reduction rate were rapidly decreased by 100 mg/L of Cr (VI). The toxic and mutagenic effects of chromium on bacteria have been reported to occur at concentration of 10–12 mg/L Cr (VI), which significantly
affected cell growth of Bacillus sp. [3]. These toxic effects are attributed to alteration of genetic material and altered physiological and metabolic reactions [31].
Fig. 6. Effect of temperature on Cr (VI) reduction of Bacillus sp. MDS05. The Cr (VI) reduction activity was measured after incubation for 24 h at initial pH 8. Error bars represent standard deviation (n 1).
5. Conclusions Microorganisms with the ability to tolerate and reduce Cr (VI) can be used for detoxification of environment contaminated with Cr (VI). In this study, we report the isolation and screening of eight Cr (VI)resistant bacterial isolates and the characterization of Cr (VI) reduction. Isolate MDS05 that exhibited more resistance to different heavy metals and substantial reduction of Cr (VI) was further studied. The factors that affected Cr(VI) reduction studied here (pH, temperature, and Cr concentration) are important environmental factors regulating remediation strategies for chromate pollution. Further investigations are underway for evaluation of Cr (VI) reduction by isolate MDS05 under various contaminated environments. Acknowledgements This work was supported by a grant from Key Natural Science Foundation of South-Central University for Nationalities (YZZ06013). References [1] K. Chourey, M.R. Thompson, J. Morrell-Falvey, N.C. VerBerkmoes, S.D. Brown, M. Shah, J. Zhou, M. Doktycz, R.L. Hettich, D.K. Thompson, Global molecular and morphological effects of 24-hour chromium(vi) exposure on Shewanella oneidensis MR-1, Appl. Environ. Microbiol. 72 (2006) 6331–6344. [2] F.A.O. Camargo, F.M. Bento, B.C. Okeke, W.T. Frankenberger, Chromate reduction by chromium-resistant bacteria isolated from soils contaminated with dichromate, J. Environ. Qual. 32 (2003) 1228–1233. [3] Y. Liu, W. Xu, G. Zeng, X. Li, H. Gao, Cr (VI). reduction by Bacillus sp. isolated from chromium landfill, Process Biochem. 41 (2006) 1981–1986. [4] S. Zafar, F. Aqil, I. Ahmad, Metal tolerance and biosorption potential of filamentous fungi isolated from metal contaminated agricultural soil, Bioresour. Technol. 98 (2007) 2557–2561. [5] S. Congeevaram, S. Dhanarani, J. Park, M. Dexilin, K. Thamaraiselvi, Biosorption of chromium and nickel by heavy metal resistant fungal and bacterial isolates, J. Hazard. Mater. 146 (2007) 270–277. [6] B. Prasenjit, S. Sumathi, Uptake of chromium by Aspergillus foetidus, J. Mater. Cycles Waste Manag. 7 (2005) 88–92. [7] A.H. Alvarez, R. Moreno-Sanchez, C. Cervantes, Chromate efflux by means of the chrA chromate resistance protein from Pseudomonas aeruginosa, J. Bacteriol. 181 (1999) 7398–7400. [8] L.H. Bopp, H.L. Ehrlich, Chromate resistance and reduction in Pseudomonas fluorescence strain LB300, Arch. Microbiol. 150 (1988) 426–431. [9] C. Garbisu, I. Alkorta, M.J. Llama, J.L. Serra, Aerobic chromate reduction by Bacillus subtilis, Biodegradation 9 (1998) 133–141. [10] P.C. Wang, T. Mori, K. Komori, M. Sasatsu, K. Toda, H. Ohtake, Isolation and characterization of an Enterobacter cloacae strain that reduces hexavalent chromium under anaerobic conditions, Appl. Environ. Microbiol. 55 (1989) 1665–1669. [11] D.P. Clark, Chromate reductase activity of Enterobacter aerogenes is induced by nitrite, FEMS Microbiol. Lett. 122 (1994) 233–238.
G. Cheng, X. Li / European Journal of Soil Biology 45 (2009) 483–487 [12] D.F. Ackerley, C.F. Gonzalez, M. Keyhan, R. Blake, A. Matin, Mechanism of chromate reduction by the Escherichia coli protein, NfsA, and the role of different chromate reductases in minimizing oxidative stress during chromate reduction, Environ. Microbiol. 6 (2004) 851–860. [13] S. Viamajala, B.M. Peyton, J.N. Petersen, Modeling chromate reduction in Shewanella oneidensis MR-1: development of a novel dual-enzyme kinetic model, Biotechnol. Bioeng. 83 (2003) 790–797. [14] H. Guha, K. Jayachandran, F. Maurrasse, Kinetics of chromium (VI) reduction by a type strain Shewanella alga under different growth conditions, Environ. Pollut. 115 (2001) 209–218. [15] Y.N. H-Holman, D.L. Perry, M.C. Martin, G. Lamble, W.R. McKinney, J.C. HunterCevera, Real-time characterization of biogeochemical reduction of Cr (VI) on basalt surfaces by SR-FTIR imaging, Geomicrobiol. J. 16 (1999) 307–323. [16] C. Michel, M. Brugma, C. Aubert, A. Bernadac, M. Bruschi, Enzymatic reduction of chromate: comparative studies using sulfate-reducing bacteria, Appl. Microbiol. Biotechnol. 55 (2001) 95–100. [17] S. Kumar, K. Tamara, I.B. Jakobsen, M. Neim, MEGA2: molecular evolutionary genetic analysis software, Bioinformatics 17 (2001) 1244–1245. [18] M.T. Hassan, D. van der Lelie, D. Springael, U. Romling, N. Ahmed, M. Mergeay, Identification of a gene cluster, czr, involved in cadmium and zinc resistance in pseudomonas aeruginosa, Gene 238 (1999) 417–425. [19] A. Hernandez, R.P. Mellado, J.L. Martinez, Metal accumulation and vanadiuminduced multidrug resistance by environmental isolates of Escherichia hermannii and Enterobacter cloacae, Appl. Environ. Microbiol. 64 (1998) 4317–4320. [20] C.H. Park, M. Keyhan, B. Wielinga, S. Fendorf, A. Matin, Purification to homogeneity and characterization of a novel Pseudomonas putida chromate reductase, Appl. Environ. Microbiol. 66 (2000) 1788–1795.
487
[21] A. Hassen, N. Saidi, M. Cherif, A. Baudabous, Effects of heavy metals on Pseudomonas aeruginosa and Bacillus huringiensis, Bioresour. Technol. 65 (1998) 73–82. [22] E.I. Yilmaz, Metal tolerance and biosorption capacity of Bacillus circulans strain EB1, Res. Microbiol. 154 (2003) 409–415. [23] M.A. Amoozegar, A. Ghasemi, M.R. Razavi, S. Naddaf, Evaluation of hexavalent chromium reduction by chromate-resistant moderately halophile, Nesterenkonia sp. strain MF2, Process Biochem. 42 (2007) 1475–1479. [24] M. Megharaj, S. Avudainayagam, R. Naidu, Toxicity of hexavalent chromium and its reduction by bacteria isolated from soil contaminated with tannery waste, Curr. Microbiol. 47 (2004) 51–54. [25] R. Elangovan, S. Abhipsa, B. Rohit, P. Ligy, K. Chandraraj, Reduction of Cr(VI) by a Bacillus sp, Biotechnol. Lett. 28 (2006) 247–252. [26] A.R. Shakoori, M. Makhdoom, R.U. Haq, Hexavalent chromium reduction by a dichromate-resistant gram-positive bacterium isolate from effluents of tanneries, Appl. Microbiol. Biotechnol. 53 (2000) 348–351. [27] A. Pal, A.K. Paul, Aerobic chromate reduction by chromate-resistant bacteria isolated from serpentine soil, Microbiol. Res. 159 (2005) 347–354. [28] Y.T. Wang, C. Xiao, Factors affecting hexavalent chromium reduction in pure cultures of bacteria, Wat. Res. 29 (1995) 2467–2474. [29] S. Sultan, S. Hasnain, Reduction of toxic hexavalent chromium by Ochrobactrum intermedium strain SDCr-5 stimulated by heavy metals, Bioresour. Technol. 98 (2007) 340–344. [30] P. Wang, T. Mori, K. Toda, H. Ohtake, Membrane-associated chromate reductase activity from Enterobacter cloacae, J. Bacteriol. 172 (1990) 1670–1672. [31] M.E. Losi, C. Amrhein, W.T. Frankenberger, Environmental biochemistry of chromium, Rev. Environ. Contam. Toxicol. 36 (1994) 91–121.
Contents lists available at ScienceDirect
European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi
Original article
Bioreduction of chromium (VI) by Bacillus sp. isolated from soils of iron mineral area Guojun Cheng*, Xiaohua Li Key Lab for Microorganism and Biological Transformation, College of Life Science, South-Central University for Nationalities, 430074 Wuhan, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 June 2008 Received in revised form 24 January 2009 Accepted 19 June 2009 Available online 7 August 2009 Handling editor: Hermann Verhoef
Extensive use of chromium in industry has caused environmental contamination. Chromium-resistant bacteria are capable of reducing toxic Cr (VI) to less toxic Cr (III). Eight isolates, which can grow on LB agar containing 500 mg/L of Cr (VI), were isolated from soil samples of iron mineral area. The bacterial isolates were identified as Bacillus sp. by the 16S rRNA gene sequences. Phylogenetic tree analysis indicates the isolates can be divided into two groups. The bacterial isolates can be resistant to other heavy metals and reduce Cr (VI) at different levels. One bacterial isolate (MDS05), which can tolerate 2500 mg/L Cr (VI) and was able to reduce almost 100% of Cr (VI) at the concentration of 10 mg/L in 24 h, was selected to study the effects of some environmental factors such as pH, temperature, and time on Cr (VI) reduction and growth. The cell growth of MDS05 was affected by the presence of Cr (VI), especially at the concentration of 100 mg/ L. It reduced more amount of Cr (VI) under a wide range of concentrations from 5 to 50 mg/L, and reduction was optimum at 37 C and pH 8. MDS05 showed great promise for use in Cr (VI) detoxification under a wide range of environmental conditions. Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords: Bacillus sp. Cr (VI) reduction Metal resistance Bioremediation
1. Introduction Chromate has many industrial applications and defense applications such as stainless steel, chrome plating, leather tanning, dyes, pigments and nuclear weapons production, and often causes a widespread environmental contaminant [1]. It generally exists in two stable oxidation states, trivalent or chromium III and hexavalent or chromium VI. The trivalent chromium is less toxic and mobile, while the hexavalent form of chromium, Cr (VI), is highly soluble and a strong oxidizing agent that is reduced intracellularly to Cr5þ and reacts with nucleic acids and other cell components to produce mutagenic and carcinogenic effects on biological systems [2]. Hence, reduction of Cr (VI) to Cr (III) is an attractive and useful method for Cr (VI) pollution [3]. Conventional physicochemical methods for removing toxic CrO24 include electrochemical treatment, precipitation, ion exchange, evaporation, reverse osmosis, and adsorption on activated coal. However, most of these methods are often inefficient and very expensive especially for metals at low concentration [2,4], and hence biological approach has been considered as an alternative remediation for chromium(VI) contamination due to the lower costs and the significant smaller quantities of the produced sludge [5].
* Corresponding author. Fax: þ86 27 67842689. E-mail address: [email protected] (G. Cheng). 1164-5563/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejsobi.2009.06.009
Biological approach could be initiated by the microbial reduction of Cr (VI). Microbial populations in Cr (VI) polluted environments adapt to toxic concentrations of Cr (VI) and become chromium (VI)-resistant and chromium (VI)-reducing strains [6]. In previous researches, a number of microorganisms have been reported to be able to reduce Cr (VI), including strains of Pseudomonas [7,8], Bacillus [9], Enterobacter [10,11], Escherichia coli [12], Shewanella [13,14], and several other bacterial isolates [15,16]. The objectives of this study was to isolate bacteria from iron mineral area where the hexavalent chromium and other heavy metal level is quite high, to measure the bacterial minimum inhibitory concentrations (MIC) of heavy metals, and to research the characterization of factors involved in hexavalent chromium reduction. These results obtained in this study may provide the useful knowledge for the bioremediation of chromate pollution. 2. Materials and methods 2.1. Soil samples and isolation of bacteria Soil samples were collected from iron mineral area in HuBei Province, China that proved many minerals such as iron, manganese, chromium and copper. For the isolation of bacteria, soil samples were serially diluted and plated onto Luria–Bertani (LB) agar. The molten medium was supplemented with Cr (VI) as K2CrO4 to final concentration 500 mg/L [2]. The Cr(VI) stock solution was
484
G. Cheng, X. Li / European Journal of Soil Biology 45 (2009) 483–487
filter sterilized with a 0.22 mm membrane filter. Plates were incubated at 37 C for 48 h. Colonies of different morphologies were then selected on LB agar containing 500 mg/L of Cr (VI) and incubated at 37 C for 24 h. 2.2. Identification of chromium-resistant bacteria The bacterial isolates were identified by 16S rRNA gene as follows. Bacterial strains were grown in LB broth supplemented with 500 mg/L of Cr (VI) at 37 C for 48 h and DNA was extracted and purified according to the method of Asubel et al. [7]. The 16S rRNA gene was PCR amplified from the isolated DNA with the following primers of 16S-L (5’AAGGAGGTGATCCAGCC3’) and 16S-R (5’AGAGTTTGATCCTGGCT CAG3’). The thermocycling conditions were as follows: 3 min at 94 C; followed by 30 cycles of 1 min at 94 C, 1 min at 56 C, and 2 min at 72 C; followed by 10 min at 72 C and a hold at 4 C. PCR products were obtained and cloned into plasmid vector pMD-18T (Tarkara, DaLian, China) and then sequenced. The nucleotides of 16S rRNA gene were carried out using BLAST (http://www.ncbi.nlm.nih.gov/BLAST). Phylogenetic tree of small Chromium-Resistant Bacteria based on 16S rRNA genes was constructed using the minimum-evolution method of Mega 4.0 software [17]. 2.3. Determination of minimum inhibitory concentrations (MIC) of heavy metals The medium used for MIC measurement was LB agar medium containing heavy metal salts in varying concentrations ranging from 50 to 3000 mg/L. The plates were checked for growth after 3 d incubation at 37 C [18]. The MIC was defined as the lowest metal concentration that completely inhibited growth. The following metal salts were used: K2CrO4, CdCl2$2.5H2O, ZnSO4$7H2O, NiSO4, CuSO4$5H2O, MnCl2$4H2O, and Pb(C2H3O2)$3H2O. 2.4. Characterization of chromium reduction by isolates Chromate-resistant bacterial isolates were inoculated into LB broth (pH 7.0) containing 10 mg/L of Cr (VI) and incubated for 24 h at 37 C with orbital shaking at 200 rpm [3]. Bacterial cell density of the liquid cultures was routinely monitored by measuring optical density (OD) at 600 nm. Bioreduction of Cr (VI) was done according to the procedure of Hernandez et al. [19]. Hexavalent chromium was determined spectrophotometrically at a wavelength of 540 nm with the 1,5-diphenylcarbazide (DPC) method [20]. 2.5. Effect of pH and temperature on chromate reduction The influence of temperature and pH on bacterial chromate reduction was assessed. For the effect of pH, the pH value of the autoclaved culture medium was adjusted to 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 with predetermined amounts of filter-sterilized 10% (w/v) NaOH and 10% (w/v) HCl at 37 C. Incubation temperature was varied at 20, 30, 37, 40 and 45 C. Bacterial isolates were inoculated at an initial optical density at 600 nm (OD600) of 0.08–0.17 into fresh LB medium containing 10 mg/L of Cr (VI).
Table 1 Identification of chromium-resistant bacteria using 16S rRNA gene sequence. Isolates
Accession no.
Similarity
Species as close relatives
MDS01 MDS02 MDS03 MDS04 MDS05 MDS06 MDS07 MDS08
EU236670 EU236671 EU236672 EU236674 EU236673 EU236675 EU236676 EU236677
99% 96% 97% 97% 99% 97% 97% 98%
Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus
indicus badius megaterium flexus megaterium thuringiensis megaterium luciferensis
PCR amplification of the 16S rRNA genes, which were then sequenced, and the nucleotide sequences were analyzed for homology using BlastN (Table 1). The results indicated that the eight 16S rRNA genes all showed more than 96% sequence homology to those in the genus Bacillus. This may imply that Bacillus sp. can tolerate the toxicity of Cr (VI) and probably have become the dominant strains in the high level of heavy metal contamination area. 3.2. Construction of phylogenetic tree A phylogenetic tree was constructed based on 16S rRNA partial sequences (Fig. 1). The results showed that the 8 isolates can be divided into two groups, the isolates MDS01, MDS06, MDS08, stood well apart from strains MDS02, MDS03, MDS04,. MDS05, MDS07. The difference in species grouping may suggest a differential rate of evolutionary divergence among the chromate-resistant Bacillus species. 3.3. Determination of heavy metal resistance All the bacterial isolates were tested for their resistance against different metal ions that are Cr6þ, Cd2þ, Zn2þ, Ni2þ, Cu2þ, Pb2þ and Mn2þ. As shown in Table 2, all isolated strains showed no growth inhibition up to 500 mg/L Cr (VI), and MDS05 even could grow with 2500 mg/L Cr (VI). The bacterial isolates also demonstrated an attractive resistance to other heavy metals (Table 2). The MIC values ranged from about 50 mg/L (Cu2þ) to 2500 mg/L (Cr6þ). From comparisons of the results across the metals, MDS05 and MDS02 exhibited a biggish MIC value of the heavy metals, and their resistance levels were considerably greater than those reported for other bacteria [19,21]. A high degree of tolerance to multiple heavy metals is necessary for the capacity of the strains to survive in different sources of pollution with elevated heavy metal levels [22]. Chromate reduction activity of whole cells was detected in all isolates at initial Cr (VI) concentration of 10 mg/L (Fig. 2). The ability of MDS02 and MDS05 to reduce Cr (VI) in 24 h period is of interest as it is relatively fast compared to other species. MDS02 and MDS05 showed a reduction of 59% and 65%, respectively, after 6 h of
3. Results 3.1. Screening and identification of bacterial isolates Eight isolates (MDS01-MDS08) with inherent ability of 500 mg/L Chromate resistance have been isolated from the soils of iron mineral area in HuBei Province, China. The isolates showed significant growth in LB medium containing 500 mg/L Cr (VI). All the isolates formed spores and were gram-positive. The isolates were identified by initial
Fig. 1. Phylogenetic tree derived from 16S rRNA gene sequences of chromium-resistant bacteria. The scale bar corresponds to 0.005 substitution per nucleotide position.
G. Cheng, X. Li / European Journal of Soil Biology 45 (2009) 483–487
485
Table 2 Resistance to heavy metals in chromium-resistant bacterial isolates. Stains
MICs (mg/L) for Bacillus. sp strains Cr6þ
Cd2þ
Zn2þ
Ni2þ
Cu2þ
Mn2þ
Pb2þ
MDS01 MDS02 MDS03 MDS04 MDS05 MDS06 MDS07 MDS08
1000 2000 1500 500 2500 500 500 1000
200 300 200 100 400 200 100 200
400 800 300 600 500 400 500 200
200 300 500 300 700 200 200 100
100 100 200 50 200 300 100 100
400 500 300 400 600 300 200 300
1000 2000 500 1500 2000 500 100 500
incubation at Cr (VI) concentration of 10 mg/L. On the next 6 h, 21% and 18%, respectively, of the added Cr (VI) was reduced, and after 24 h, more than 99% of Cr (VI) was reduced. Isolate MDS05 was selected for further studies due to the relatively higher Cr (VI) reduction and higher resistance levels of heavy metals in this strain. 3.4. Effect of Cr (VI) on cell growth The effect of Cr (VI) on the growth of MDS05 was evaluated before the experiments of Cr (VI) reduction. Fig. 3 shows MDS05 was exposed to six different Cr (VI) concentrations, and bacterial density (OD600) was measured after 24 h. It is obvious that Cr (VI) at 1, 5, 10 and 50 mg/l has slight effect on the growth of cells. However, growth was inhibited by 100 mg/L of Cr (VI) and decreased cell density by about 20%. The growth curves of MDS05 in LB medium with or without 10 mg/ l Cr (VI) is shown in Fig. 4. The growth curve of MDS05 in the medium containing Cr (VI) did not follow the same growth pattern as the control. Growth of MDS05 in the presence of 10 mg/l Cr (VI) showed a longer lag phase than that in the absence of Cr (VI). It indicates that the toxic Cr (VI) effect cell growth during the incubation time of 24 h. 3.5. Factors affecting on chromium reduction Initial pH of the medium with 10 mg/L Cr (VI) supplied was considered as a factor for Cr (VI) reduction by MDS05 (Fig. 5). Values for pH of 5 and 10 restricted bacterial Cr (VI) reduction, while at pH ranging from 7 to 9, MDS05 had high activity for Cr (VI) reduction. The optimum initial pH value was 8. As at pH 8, almost complete Cr (VI) was reduced during the incubation time of 24 h. Microbial Cr (VI) reduction was dependent on temperature. Cr (VI) reduction by MDS05 strain was investigated over four different temperatures: 20, 30, 37 and 45 C (Fig. 6). Cr (VI) was reduced effectively at 30 and 37 C, with reduction 91% and 100%, respectively.
Fig. 2. Cr (VI) reduction of Cr (VI)-resistant bacteria. The cells were cultured on Luria– Bertani agar supplemented with 10 mg/L Cr (VI). The Cr (VI) reduction activity was measured after incubation for 0, 6, 12, 18, 24 h at 37 C. Error bars represent standard deviation (n 1).
Fig. 3. Growth of Bacillus sp. MDS05. The cells were cultured on Luria–Bertani agar supplemented with 0, 1, 5, 10, 50, 100 and 300 mg/L Cr (VI), respectively. The optical density was measured after incubation for 24 h at 37 C. Error bars represent standard deviation (n 1).
While at 20 and 45 C, chromate reduction was severely decreased. This indicates that MDS05 strain reduced Cr (VI) better at 37 C. The Cr (VI)-resistant bacterial MDS05 was exposed to six different Cr (VI) concentrations, and Cr (VI) reduction was evaluated (Fig. 7). At initial concentration of 5, 10 and 50 mg/l, MDS05 strain showed the highest Cr (VI) reduction rate, while a sharp decrease was observed in presence of a high initial concentration of Cr (VI) at 100 mg/L. 4. Discussion Metal-polluted environments pose serious health and ecological risk. Metal containing industrial effluents constitute a major source of metallic pollution [23]. Soil is a common environment for Cr(VI) contamination, and most Cr (VI)-resistant bacteria reported to date have been isolated from soil. Here we describe the identification of 8 Cr(VI)-resistant bacterial isolates and the characterization of Cr(VI) reduction by selected isolate. Most of the isolates were able to reduce Cr(VI), but at different rates. The isolates all belonged to the genera Bacillus. Few isolates of the genus Bacillus have been known to tolerate and reduce Cr(VI) [9,24,25]. The tolerance level of Cr(VI) for the new Bacillus sp. MDS05 was 2500 mg/L, which is more than other strain of Bacillus sp. those have been known to tolerate no more than 2500 mg/L Cr(VI). However, a bacterial strain (probably a Bacillus species) from a tannery effluent has been reported which is resistant to very high level of Cr(VI) at more than 8 mg/mL [26].
Fig. 4. Growth curves of Bacillus sp. MDS05 in the absence or in the presence of 10 mg/L Cr (VI). Error bars represent standard deviation (n 1).
486
G. Cheng, X. Li / European Journal of Soil Biology 45 (2009) 483–487
Fig. 5. Effect of pH on Cr (VI) reduction of Bacillus sp. MDS05. Initial pH of the culture medium was adjusted to 5–10. The Cr (VI) reduction activity was measured after incubation for 24 h at 37 C. Error bars represent standard deviation (n 1).
Fig. 7. Effect of initial Cr (VI) concentration on Cr (VI) reduction of Bacillus sp. MDS05. The Cr (VI) reduction activity was measured after incubation for 24 h at pH 8 and 37 C. Error bars represent standard deviation (n 1).
The strain MDS05 was shown to be of a high efficiency in detoxifying chromate. It could rapidly reduce almost 100% of 10 mg/ L Cr(VI) with 24 h. Pal and Paul [27] reported an isolate B. sphaericus AND303 has high Cr(VI) removal efficiency, and it can removed about 72% Cr(VI) (10 mg/L) from the medium within 24 h. Liu et al. [3] also reported that Bacillus sp. XW-4 was capable of removing 100% of 10 mg/L in 24 h, but it can only tolerate 100 mg/L Cr (VI). Cr(VI) reduction were dependent on pH, temperature, and Cr concentration. The optimum pH for growth of the isolate MDS05 was 7.0–9.0. Extreme pH (5.0, 6.0 and 10.0) restricted Cr(VI) reduction. Variation of the culture initial pH highly affected Cr(VI) reduction. Camargo et al. [2] reported that optimal pH for Cr (VI) reduction by five chromate bacteria was 7–8. Wang and Xiao [28] also reported that optimal pH for Cr (VI) reduction of Bacillus sp. and Pseudomonas fluorescence was 7, but it was strongly inhibited at pH 6. The change in optimal pH indicates that pH modification is important for different cultures to achieve the maximum Cr(VI) reduction in Cr (VI) detoxification. Temperature is also an important factor for bacterial Cr(VI) reduction. The optimum of the tested temperatures was 37 C for Cr(VI) reduction of MDS05. Extreme temperature (20 and 45 C) restricted Cr(VI) reduction. Optimal temperature for Cr (VI) reduction of Cr (VI)-resistant bacteria was reported at 37 C, but it was almost ceased at 20 C [29]. Wang et al. [30] reported that no chromate reduction was observed at 4 and 60 C. Since Cr (VI) reduction is enzyme-mediated, changes in temperature will affect the enzyme activity. In addition, the results clearly showed a direct positive correlation between Cr(VI) reduction and bacterial growth with Cr(VI) concentration of 1–100 mg/L. Both growth and Cr (VI) reduction rate were rapidly decreased by 100 mg/L of Cr (VI). The toxic and mutagenic effects of chromium on bacteria have been reported to occur at concentration of 10–12 mg/L Cr (VI), which significantly
affected cell growth of Bacillus sp. [3]. These toxic effects are attributed to alteration of genetic material and altered physiological and metabolic reactions [31].
Fig. 6. Effect of temperature on Cr (VI) reduction of Bacillus sp. MDS05. The Cr (VI) reduction activity was measured after incubation for 24 h at initial pH 8. Error bars represent standard deviation (n 1).
5. Conclusions Microorganisms with the ability to tolerate and reduce Cr (VI) can be used for detoxification of environment contaminated with Cr (VI). In this study, we report the isolation and screening of eight Cr (VI)resistant bacterial isolates and the characterization of Cr (VI) reduction. Isolate MDS05 that exhibited more resistance to different heavy metals and substantial reduction of Cr (VI) was further studied. The factors that affected Cr(VI) reduction studied here (pH, temperature, and Cr concentration) are important environmental factors regulating remediation strategies for chromate pollution. Further investigations are underway for evaluation of Cr (VI) reduction by isolate MDS05 under various contaminated environments. Acknowledgements This work was supported by a grant from Key Natural Science Foundation of South-Central University for Nationalities (YZZ06013). References [1] K. Chourey, M.R. Thompson, J. Morrell-Falvey, N.C. VerBerkmoes, S.D. Brown, M. Shah, J. Zhou, M. Doktycz, R.L. Hettich, D.K. Thompson, Global molecular and morphological effects of 24-hour chromium(vi) exposure on Shewanella oneidensis MR-1, Appl. Environ. Microbiol. 72 (2006) 6331–6344. [2] F.A.O. Camargo, F.M. Bento, B.C. Okeke, W.T. Frankenberger, Chromate reduction by chromium-resistant bacteria isolated from soils contaminated with dichromate, J. Environ. Qual. 32 (2003) 1228–1233. [3] Y. Liu, W. Xu, G. Zeng, X. Li, H. Gao, Cr (VI). reduction by Bacillus sp. isolated from chromium landfill, Process Biochem. 41 (2006) 1981–1986. [4] S. Zafar, F. Aqil, I. Ahmad, Metal tolerance and biosorption potential of filamentous fungi isolated from metal contaminated agricultural soil, Bioresour. Technol. 98 (2007) 2557–2561. [5] S. Congeevaram, S. Dhanarani, J. Park, M. Dexilin, K. Thamaraiselvi, Biosorption of chromium and nickel by heavy metal resistant fungal and bacterial isolates, J. Hazard. Mater. 146 (2007) 270–277. [6] B. Prasenjit, S. Sumathi, Uptake of chromium by Aspergillus foetidus, J. Mater. Cycles Waste Manag. 7 (2005) 88–92. [7] A.H. Alvarez, R. Moreno-Sanchez, C. Cervantes, Chromate efflux by means of the chrA chromate resistance protein from Pseudomonas aeruginosa, J. Bacteriol. 181 (1999) 7398–7400. [8] L.H. Bopp, H.L. Ehrlich, Chromate resistance and reduction in Pseudomonas fluorescence strain LB300, Arch. Microbiol. 150 (1988) 426–431. [9] C. Garbisu, I. Alkorta, M.J. Llama, J.L. Serra, Aerobic chromate reduction by Bacillus subtilis, Biodegradation 9 (1998) 133–141. [10] P.C. Wang, T. Mori, K. Komori, M. Sasatsu, K. Toda, H. Ohtake, Isolation and characterization of an Enterobacter cloacae strain that reduces hexavalent chromium under anaerobic conditions, Appl. Environ. Microbiol. 55 (1989) 1665–1669. [11] D.P. Clark, Chromate reductase activity of Enterobacter aerogenes is induced by nitrite, FEMS Microbiol. Lett. 122 (1994) 233–238.
G. Cheng, X. Li / European Journal of Soil Biology 45 (2009) 483–487 [12] D.F. Ackerley, C.F. Gonzalez, M. Keyhan, R. Blake, A. Matin, Mechanism of chromate reduction by the Escherichia coli protein, NfsA, and the role of different chromate reductases in minimizing oxidative stress during chromate reduction, Environ. Microbiol. 6 (2004) 851–860. [13] S. Viamajala, B.M. Peyton, J.N. Petersen, Modeling chromate reduction in Shewanella oneidensis MR-1: development of a novel dual-enzyme kinetic model, Biotechnol. Bioeng. 83 (2003) 790–797. [14] H. Guha, K. Jayachandran, F. Maurrasse, Kinetics of chromium (VI) reduction by a type strain Shewanella alga under different growth conditions, Environ. Pollut. 115 (2001) 209–218. [15] Y.N. H-Holman, D.L. Perry, M.C. Martin, G. Lamble, W.R. McKinney, J.C. HunterCevera, Real-time characterization of biogeochemical reduction of Cr (VI) on basalt surfaces by SR-FTIR imaging, Geomicrobiol. J. 16 (1999) 307–323. [16] C. Michel, M. Brugma, C. Aubert, A. Bernadac, M. Bruschi, Enzymatic reduction of chromate: comparative studies using sulfate-reducing bacteria, Appl. Microbiol. Biotechnol. 55 (2001) 95–100. [17] S. Kumar, K. Tamara, I.B. Jakobsen, M. Neim, MEGA2: molecular evolutionary genetic analysis software, Bioinformatics 17 (2001) 1244–1245. [18] M.T. Hassan, D. van der Lelie, D. Springael, U. Romling, N. Ahmed, M. Mergeay, Identification of a gene cluster, czr, involved in cadmium and zinc resistance in pseudomonas aeruginosa, Gene 238 (1999) 417–425. [19] A. Hernandez, R.P. Mellado, J.L. Martinez, Metal accumulation and vanadiuminduced multidrug resistance by environmental isolates of Escherichia hermannii and Enterobacter cloacae, Appl. Environ. Microbiol. 64 (1998) 4317–4320. [20] C.H. Park, M. Keyhan, B. Wielinga, S. Fendorf, A. Matin, Purification to homogeneity and characterization of a novel Pseudomonas putida chromate reductase, Appl. Environ. Microbiol. 66 (2000) 1788–1795.
487
[21] A. Hassen, N. Saidi, M. Cherif, A. Baudabous, Effects of heavy metals on Pseudomonas aeruginosa and Bacillus huringiensis, Bioresour. Technol. 65 (1998) 73–82. [22] E.I. Yilmaz, Metal tolerance and biosorption capacity of Bacillus circulans strain EB1, Res. Microbiol. 154 (2003) 409–415. [23] M.A. Amoozegar, A. Ghasemi, M.R. Razavi, S. Naddaf, Evaluation of hexavalent chromium reduction by chromate-resistant moderately halophile, Nesterenkonia sp. strain MF2, Process Biochem. 42 (2007) 1475–1479. [24] M. Megharaj, S. Avudainayagam, R. Naidu, Toxicity of hexavalent chromium and its reduction by bacteria isolated from soil contaminated with tannery waste, Curr. Microbiol. 47 (2004) 51–54. [25] R. Elangovan, S. Abhipsa, B. Rohit, P. Ligy, K. Chandraraj, Reduction of Cr(VI) by a Bacillus sp, Biotechnol. Lett. 28 (2006) 247–252. [26] A.R. Shakoori, M. Makhdoom, R.U. Haq, Hexavalent chromium reduction by a dichromate-resistant gram-positive bacterium isolate from effluents of tanneries, Appl. Microbiol. Biotechnol. 53 (2000) 348–351. [27] A. Pal, A.K. Paul, Aerobic chromate reduction by chromate-resistant bacteria isolated from serpentine soil, Microbiol. Res. 159 (2005) 347–354. [28] Y.T. Wang, C. Xiao, Factors affecting hexavalent chromium reduction in pure cultures of bacteria, Wat. Res. 29 (1995) 2467–2474. [29] S. Sultan, S. Hasnain, Reduction of toxic hexavalent chromium by Ochrobactrum intermedium strain SDCr-5 stimulated by heavy metals, Bioresour. Technol. 98 (2007) 340–344. [30] P. Wang, T. Mori, K. Toda, H. Ohtake, Membrane-associated chromate reductase activity from Enterobacter cloacae, J. Bacteriol. 172 (1990) 1670–1672. [31] M.E. Losi, C. Amrhein, W.T. Frankenberger, Environmental biochemistry of chromium, Rev. Environ. Contam. Toxicol. 36 (1994) 91–121.