Removal of Toxic Chromate Using Free and Immobilized Cr(VI)-Reducing Bacterial Cells of Intrasporangium Sp. Q5-1

World J Microbiol Biotechnol DOI 10.1007/s11274-009-0047-x

ORIGINAL PAPER

Removal of toxic chromate using free and immobilized Cr(VI)-reducing bacterial cells of Intrasporangium sp. Q5-1

Jinxia Yang Æ Minyan He Æ Gejiao Wang

Received: 11 February 2009 / Accepted: 8 April 2009 Ó Springer Science+Business Media B.V. 2009

Abstract Chromate-reducing microorganisms with the Introduction ability of reducing toxic chromate [Cr(VI)] into insoluble trivalent chromium [Cr(III)] are very useful in treatment of The wide use of chromium (Cr) in industries such as leather Cr(VI)-contaminated water. In this study, a novel chro- tanning, metallurgy, electroplating, textile, and pigment mate-reducing bacterium was isolated from Mn/Cr-con- manufacturing has resulted in large quantities of chromium- taminated soil. Based on morphological, physiological/ containing industrial effluent in China and in the World biochemical characteristics and 16S rRNA gene sequence (Wang and Xiao 1995; Pattanapipitpaisal et al. 2001; Sultan analyses, this strain was identified as Intrasporangium sp. and Hasnain 2007). Haxavalent chromium [chromate, strain Q5-1. This bacterium has high Cr(VI) resistance with Cr(VI)] and trivalent chromium Cr(III) are the most com- a MIC of 17 mmol l-1 and is able to reduce Cr(VI) aero- mon oxidation states (Megharaj et al. 2003). Cr(VI) is highly bically. The best condition of Cr(VI) reduction for Q5-1 is toxic, mobile and soluble, which generally exists as an 2- 2- pH 8.0 at 37°C. Strain Q5-1 is also able to reduce Cr(VI) in oxyanion (CrO4 ) in aqueous systems. CrO4 is a strong resting (non-growth) conditions using a variety of carbon oxidizing agent, which reacts with nucleic acids and other sources as well as in the absence of a carbon source. cell components and results in toxicity, mutants and carci- Acetate (1 mmol l-1) is the most efficient carbon source nogenesis (Clark 1994; Codd et al. 2001; Ackerley et al. for stimulating Cr(VI) reduction. In order to apply strain 2006). Cr(VI) has been placed as a priority pollutant, and Q5-1 to remove Cr(VI) from wastewater, the bacterial cells classified as a class A human carcinogen by the US Envi- were immobilized with different matrices. Q5-1 cells ronmental Protection Agency (USEPA; Cieslak-Golonka embedded with compounding beads containing 4% PVA, 1995; Costa and Klein 2006). Cr(III), on the other hand, is 3% sodium alginate, 1.5% active carbon and 3% diatomite insoluble and less toxic (Mclean and Beveridge 2000; Upreti showed a similar Cr(VI) reduction rates to that of free cells. et al. 2004). Therefore reduction of Cr(VI) to Cr(III) is an In addition, the immobilized Q5-1 cells have the advanta- effective way for remediation of Cr(VI)-polluted water. ges over free cells in being more stable, easier to re-use and Conventional technologies for Cr(VI)-contaminated minimal clogging in continuous systems. This study pro- wastewater treatments include chemical reduction followed vides potential applications of a novel immobilized chro- by precipitation, ion exchange, and adsorption on alum or mate-reducing bacterium for Cr(VI) bioremediation. kaolinite etc. However, most of these technologies need high energy or large input of chemical reagents that may Keywords Bioremediation Bacterial immobilization cause secondary environmental pollutions (Komori et al. Cr Chromate-reducing bacterium Intrasporangium 1990). Alternatively, an increasing attention has been paid to use bioremediation approaches through selective microorganisms that capable of reducing Cr(VI) to the less J. Yang M. He G. Wang (&) toxic and insoluble Cr(III). State Key Laboratory of Agricultural Microbiology, College of Reduction of Cr(VI) has been reported in several bac- Life Science and Technology, Huazhong Agricultural University, 430070 Wuhan, People’s Republic of China teria under aerobic or anaerobic conditions, including e-mail: [email protected]; [email protected] Bacillus (Campos et al. 1995; Camargo et al. 2003b; 123 World J Microbiol Biotechnol

Elangovan et al. 2006; Liu et al. 2006; Desai et al. 2008), county, Hunan province, P. R. China. This region is highly Pseudomonas (Salunkhe et al. 1998;Parketal.2000; heavy metal contaminated with up to 3.27 g kg-1 Mn in Mclean and Beveridge 2000; Ganguli and Tripathi 2002), the soil (Guo et al. 2006). For enrichment, 100 g soil

Escherichia coli (Bae et al. 2004), Microbacterium (Pat- sample was amended with K2CrO4 at a ﬁnal concentration tanapipitpaisal et al. 2001) and Shewanella (Myers et al. of 5 mM and kept in dark at 37°C for 1 week. Cr(VI) 2000; Vaimajala et al. 2002) etc. In the presence of oxygen, resistant bacteria were isolated from the soil sample by microbial reduction of Cr(VI) is catalyzed by soluble adding 10 g soil to 100 ml of 0.85% NaCl solution and enzymes (Cervantes and Campos 2007). Under anaerobic shaking at room temperature for 10 min. The extraction conditions, both soluble and membrane associated enzymes solution was serially diluted and plated onto Luria Broth of the electron transfer system were reported to associate (LB) plates (10 g tryptone, 10 g NaCl, 5 g yeast extract in

Cr(VI) reduction that coupled to the oxidation of an elec- 1 l distilled water) containing 2 mM K2CrO4. The plates tron donor substrate. In this process, Cr(VI) serves as the were incubated at 37°C for 1 week. Single colonies were terminal electron acceptor of an electron transfer chain that re-streaked several times to obtain pure isolates. Bacterial frequently involves cytochrome b/c (Cervantes and Cam- chromate resistant level was checked by inoculating 1% pos 2007). The Cr(III) species forms an insoluble precipi- original culture of the pure isolate (OD600 = 1.0) into LB tate, such as Cr(OH)3, which can be removed from medium that amended with different concentrations of wastewater (Jeyasingh and Philip 2005). K2CrO4. The growth of each treatment was observed after Chromate-reducing bacteria have been used in labora- incubation at 37°C for 3 days. The MIC, defined as the tory study for bacterial chromate removal from water lowest K2CrO4 concentration that completely inhibits the system (Camargo et al. 2003a; Desai et al. 2008). However, growth of strain Q5-1, was determined (Sarangi and for industrial purposes, using free bacterial cells is disad- Krishnan 2007). vantageous due to the difficulty of biomass/effluent sepa- Quantitative characterization of bacterial Cr(VI) reduc- ration (White et al. 1995) etc. These problems may be tion abilities was carried out under an aerobic condition in overcome by the use of immobilized bacterial cells with 250 ml culture flasks containing 50 ml LB medium. The the advantages of stability, regeneration, solid–liquid sep- flasks with the LB medium was supplemented with 1 mM aration and minimal clogging in continuous systems (Po- K2CrO4 and inoculated with 1% bacterial culture -1 opal and Laxman 2008). Cell immobilization has been (OD600 = 1.0) and incubated at 37°C with 160 rev min accomplished using a variety of supporting materials such shaking for up to 2 days. Controls without bacterial inoc- as natural (agar, alginate, active carbon, and diatomite etc.) ulation were also incubated in the same condition to and synthetic matrices (polyacrylamide, polyethylene gly- monitor the abiotic Cr(VI) reduction. The samples were col, and polyvinyl alcohol etc.; Seung et al. 2005). The aseptically taken at about every 4 h, centrifuged at choice of immobilization materials is different with various 6,000 rev min-1 for 10 min. Cr(VI) concentration in the microorganisms. Combination parameter of supporting supernatant was measured using 1,5-diphenyl carbazide matrices is an also key factor of an immobilized biocatalyst (DPC) reagent at absorbance value of 540 nm using a UV (Poopal and Laxman 2008). spectrophotometer (DU800, Beckman, CA, USA; APHA The objectives of this research were to: (1) isolate a 1995). A standard curve was generated using 0.2, 0.8, 1.0, novel chromate-reducing bacterium and study its chromate 1.5, and 2 mM K2CrO4. The growth of the bacterium was removal efficiency. Bacterial identification was performed determined at the Optimal Density of 600 nm (OD600). using morphological, biochemical/biophysical and 16S rRNA gene analyses; (2) evaluate the Cr(VI) reducing Morphological and biochemical/biophysical analyses abilities using growing, resting, and immobilized chro- of the Cr(VI)-reducing bacterium mate-reducing bacterial cells. The immobilized bacterium may provide a superior potential in bioremediation of Colony morphologies of the bacterium were observed on chromate pollution in various environments. LB plates after incubating at 37°C for 3 days. Cell morphologies were examined under a JSM-6390/LV scanning electron microscope (SEM; JSM-6390, JEOL, Japan) with Materials and methods 20,000 V accelerating voltage and 10,000 times enlarge- ment. Gram staining was performed using bacterial colo- Sample collection and isolation of Cr(VI)-reducing nies on LB plates as described by Bailey and Scott (1966). bacteria Biochemical and biophysical analyses were performed according to the Bergey’s manual (Holt et al. 1994). Soil sample was collected from the surface horizon Characteristics of catalase and oxidase activities, hydroly- (0–15 cm) in a manganese/chromium mine in Huayuan sis of starch, gelatin liquefaction, Voges-Proskauer (V.P.) 123 World J Microbiol Biotechnol

and Methyl Red (M.R.) reactions, indole production, H2S In a repeated-adding of Cr(VI) aliquot experiment, production and the utilization of sole carbon/nitrogen 250 ml LB (pH 8.0) was amended with 1 mM K2CrO4 and sources were tested. incubated as described above. Cr(VI) reduction was mon- itored at about 8 h time interval. After Cr(VI) reduction

16S rRNA gene identiﬁcation, DNA sequencing was nearly complete, 1 mM K2CrO4 was added into the and phylogenetic analysis culture again (a total of four times).

DNA of the bacterial strain was extracted using standard Cr(VI) reduction by resting cells of strain Q5-1 methods (Sambrook and Russell 2001). The nearly and detection of the effects of different carbon full-length 16S rRNA gene was amplified by PCR using sources on bacterial Cr(VI) reduction universal primers 27F (50-AGAGTTTGATCCTGGCT CAG-30) and 1492R (50-GGTTACCTTGTTACGACTT-30; For these experiments, bacterial cells of Intrasporangium sp. Wilson et al. 1990). PCR amplification was performed in a Q5-1 that grew overnight (18 h) in 400 ml LB (without -1 50 ll volume containing 10 ng DNA, 10 ng of each pri- K2CrO4) was harvested by centrifugation (6,000 rev min mer, 200 lM of each dNTP, 2.5 mM MgCl2,5llof109 for 20 min at 4°C), washed twice with 10 mM Tris–HCl (pH PCR buffer (100 mM Tris–HCl (pH 8.3), 100 mM KCl) 8.0), resuspended with 100 ml of the same Tris–HCl buffer and 2.5 units of Taq DNA polymerase (Fermentas, Hano- and amended with 0.2 mM K2CrO4. Each 10 ml of this ver, MD, USA). The PCR program consisted of an initial bacterial suspension was added with a carbon source [glu- 5 min denaturation step at 94°C; 30 cycles of 45 s at 94°C, cose (0.5 mM), lactose (0.5 mM), sucrose (0.5 mM), 45 s at 49°C, 1.5 min at 72°C; and a final 5 min extension methanol (1.0%, v/v), ethanol (1.0%, v/v), acetate (0.5 mM) step at 72°C. or NADH (Nicotinamide adenine dinucleotide-reduced The PCR products were separated on a 1% agarose gel disodium salt-trihydrate, 0.4 mM)], and incubated at 37°C and purified using the UltraPureTM PCR Kit (SBS Genetech, for up to 6 h as described above. Heat killed bacterial cells Beijing, China). DNA sequencing analysis was performed (100°C, 10 min) were used as a control. After incubation, using ABI 3730XL DNA analyzer by SUNBIO Company the solutions were centrifuged (6,000 rev min-1 for 20 min (Beijing, China). Sequences were analyzed by BlastN at 4°C) and Cr(VI) concentration was measured as described searching tools (http://www.ncbi.nlm.nih.gov/blast). The above. sequences were edited for the same lengths and compared using ClustalX 1.83 software (Thompson et al. 1997). A Bacterial immobilization with different embedding phylogenetic tree was constructed using the neighbor-join- matrix combinations ing distance method with the MEGA 3.1 software (http://www.megasoftware.net; Kumar et al. 2004) and the Different concentration of natural materials (sodium algi- reliability of the inferred tree was tested with 1,000 boot- nate, active carbon and diatomite) and a synthetic material strap. Some reference sequences from the GenBank were [polyvinyl alcohol (PVA)] were used to immobilize strain used in generating the phylogenetic tree for clarification. Q5-1 cells to perform Cr(VI) reduction experiments (Table 2). The spherical particles was prepared as follows: Aerobic Cr(VI) reduction by growing cells (1) PVA, sodium alginate (medium viscosity), active car- of strain Q5-1 bon and diatomite were mixed in 9 ml of deionized water, and heated to 60°C to dissolve the PVA; (2) After complete The optimal conditions of the growth and Cr(VI) reduction dissolution of PVA and when the solution was cooled to efficiency of strains Q5-1 were studied including the 40°C, about 1 g (fresh weight) fresh strain Q5-1 cells (grew effects of pH, initial Cr(VI) concentration and repeated- overnight) were added and mixed; (3) Cell beads were adding of Cr(VI) aliquots. prepared by extruding the mixture as drops into the To study the optimum pH condition, LB medium con- immobilizing phase using a sterile 10 ml disposable plastic taining 1 mM K2CrO4 with different initial pH (from 6.0 to syringe with a 21-G needle into 50 ml degassed saturated 10.0) was inoculated with 1% original culture (OD600 = boric acid solution containing 2% (w/v) calcium chloride, 1.0) and incubated at 37°C with 160 rev min-1 shaking for and immersed for 24 h. Beads (3–5 mm in diameter) were up to 2 days. The Cr(VI) concentration was tested as washed three times with 200 ml sterile distilled water and described above. To test the efficiency of bacterial Cr(VI) added aseptically to 50 ml LB medium containing 0.5 mM reduction with different initial Cr(VI) concentration, liquid K2CrO4 in a 250 ml flask. The flasks were incubated at LB (pH 8.0) amended with various initial concentrations of 37°C with 160 rev min-1 shaking. Samples were taken at

K2CrO4 (2–5 mM) were studied as described in the pH regular intervals and the Cr(VI) concentration was detected experiments. as described above. 123 World J Microbiol Biotechnol

Nucleotide sequence accession number Physiological and biochemical analyses showed that

strain Q5-1 was positive for catalase and H2S production, The nearly full-length 16S rRNA gene sequence of strain and was negative for oxidase, starch/ﬁbrin hydrolyses, Q5-1 is posted in the NCBI GenBank database (http:// gelatin liquefaction, indole production, nitrate reduction, www.ncbi.nlm.nih.gov/) as FJ487951. V.P. and M.R. test. Galactose, mannose, acetate and pyruvate were utilized as sole carbon sources by strain Q5- 1, but glucose, sucrose, maltose, ribose, maltose, starch, Results xylose, trehalose, formate, ethanol, glycerol, mannitol, and myoinositol were not utilized. Isoleucine and cystein were Isolation and characterization of the chromate-reducing utilized as sole nitrogen sources, but valine, histidine, bacterium lysine, methionine, serine, alanine were not utilized (Table 1). These physiological/biochemical analyzing Chromate-reducing bacteria were isolated by an aerobic results of strain Q5-1 met the typical characteristics of cultivation using LB medium amended with Cr(VI) and Intrasporangium. analyzed the Cr(VI) reduction using a UV spectropho- After cultivation of Q5-1 for 20 h with and without tometer. A chromate-reducing bacterial strain Q5-1 was 0.5 mM K2CrO4, the Cr(VI) concentration was determined successfully isolated from the soil sample of a manganese/ and the cell morphologies were observed under a SEM at chromium mine. Q5-1 is gram-positive, rod cells when the same time. Without adding K2CrO4, the cells were grown in LB medium and showed a high Cr(VI) resistant short rods (0.5–1.5 lm), with smooth surfaces and rela- level with a MIC of 17 mM. Table 1 shows some impor- tively regular shapes (Fig. 1a). After 20 h incubation with tant characteristics of the strain. K2CrO4, the 0.5 mM K2CrO4 was reduced to 0.005 mM

Table 1 Important Characteristics Results characteristics of the chromate- reducing bacterium Q5-1 Originated from Soil Colony morphology Bright yellow, round, moist Cell morphology Average 0.5–1.5 lm, short rods Temperature optimum (°C) 37 pH optimum for growth and Cr(VI) reduction 8.0

Gram staining, catalase reaction, H2S production ? Oxidase reaction, starch hydrolysis, V.P. and M.R. test, indole - production, gelatin liquefaction, nitrate reduction and ﬁbrin hydrolysis Carbon sources (glucose, sucrose, maltose, ribose, maltose, - starch, xylose, trehalose, formate, ethanol, glycerol, mannitol and myoinositol) Carbon sources (galactose, mannose, acetate and pyruvate) ? Nitrogen sources (isoleucine and cysteine) ? Nitrogen sources (valine, histidine, lysine, methionine, serine - and alanine) Highest 16S rRNA gene identify Intrasporangium calvum (97% identity) ?, positive; -, negative

Fig. 1 SEM photographs of strain Q5-1 cells. Scale bars: 1 lm. a The Q5-1 cells that grew in LB medium for 20 h without K2CrO4. b The Q5-1 cells that grew in LB medium amended with 0.5 mM K2CrO4 for 20 h

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Fig. 2 A neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic relationship among Q5-1 and other species. GenBank accession numbers are given in parentheses. The bootstrap numbers indicate the value of 100 replicate trees supporting the branching order. Bootstrap values [55% are shown at branch points. The scale bar represents ﬁve nucleotide substitutions per 1,000 nucleotides

(99% removed) and the cells became longer rods (2.0– 6.0 to 8.0. The maximum growth and Cr(VI) reduction rate 3.0 lm) with protrusions on the cell surfaces (Fig. 1b). were all observed at pH 8.0 (data not shown). The nearly full-length 16S rRNA gene sequence of Q5-1 The Cr(VI) reduction ability of strain Q5-1 was also were obtained and used for BLAST searches in GenBank determined by adding various concentrations of Cr(VI). As and for phylogenetic analyses. The highest 16S rRNA gene shown in Fig. 3b, at an initial Cr(VI) concentration of 2, 3 sequence similarity of Q5-1 was a 97% identity with Int- or 4 mM, Cr(VI) concentration was reduced to 0.08 mM rasporangium calvum ATCC 23552T. The tree constructed (98% removed) in 36 h, 0.08 mM (98% removed) in 48 h by the NJ method based on 16S rRNA gene sequence and 0.13 mM (98% removed) in 84 h, respectively. analysis showed that strain Q5-1 belonging to the same Whereas in the initial Cr(VI) concentration of 5 mM, branch with I. calvum ATCC 23552T (Fig. 2). Cr(VI) was reduced to 1.42 mM (about 70% removed) in Based on above morphological, physiological/bio- 72 h, and in the following 18 h, Cr(VI) can not be further chemical characteristics and 16S rRNA gene sequence reduced (Fig. 3b). At initial Cr(VI) concentrations of 2, 3 comparison, this strain was identiﬁed and named as Int- or 4 mM, the growth rates of Q5-1 were nearly the same as rasporangium sp. Q5-1. the no adding one, but when the Cr(VI) concentration reached 5 mM, the growth of Q5-1 was weakly inhibited Aerobic Cr(VI) reduction by growing cells (data not shown). of strain Q5-1 Cr(VI) reduction tests using repeated addition Figure 3a shows the growth of Intrasporangium sp. Q5-1 of Cr(VI) aliquots and time course of the Cr(VI) reduction. In the ﬁrst 10 h, the rate of growth was slow. Cr(VI) reduction began at This study was performed to test the Cr(VI) reduction about 10 h when the bacterial concentration reached at ability of Q5-1 by four times repeated addition of 1 mM

OD600 of 0.2. During the log phase (12–24 h), the Cr(VI) K2CrO4. As shown in Fig. 3c, when ﬁrst adding of 1 mM reduction rate was the fastest, along with the fast growth K2CrO4, the Cr(VI) concentration was reduced to rate of the bacterium. After 24 h, almost all of the soluble 0.10 mM in 24 h, the reduction rate was 37.41 lMh-1; Cr(VI) was removed (from 0.98 mM to 0.06 mM). LB when the second aliquot of 1 mM Cr(VI) was added, medium (pH 8.0) amended with 1 mM K2CrO4 without Cr(VI) was reduced to 0.12 mM from the 24th h to the bacterial inoculation showed no obvious Cr(VI) reduction 56th h (reduction rate: 30.60 lMh-1); the third aliquot of (Fig. 3a). The Cr(VI) reduction product was insoluble 1 mM Cr(VI) was reduced to 0.19 mM from the 56th h to white precipitants in the bottom of the conical ﬂask, which the 96th h (reduction rate: 23.20 lMh-1); The fourth is most likely the Cr(OH)3 or Cr2O3 (Puzon et al. 2005). aliquot Cr(VI) was reduced from 1.19 mM to 0.71 mM from the 96th h to the 136th h, with the reduction rate Effect of pH and initial Cr(VI) concentration decreased to 12.10 lMh-1. After 136 h, the Cr(VI) on Cr(VI) reduction reduction ability of strain Q5-1 became lower and lower. Overall, the result showed that strain Q5-1 was able to Intrasporangium sp. Q5-1 grew in a pH range from 6.0 to reduce Cr(VI) without addition of any extra nutrients 10.0. Chromate reduction was observed in a pH range from within about 136 h. 123 World J Microbiol Biotechnol

NADH, methanol and ethanol did not show the enhance- ment of Cr(VI) reduction. However, the resting cells of Q5- 1 (previously grew in LB medium) without adding any additional carbon source still showed an efﬁcient Cr(VI) reduction in 6 h (Fig. 4;CK?). The heat killed bacterial resting cells showed non obvious Cr(VI) reduction (Fig. 4; CK-). Overall, the difference of reduction concentration was not very signiﬁcant with or without any carbon source, except for acetate (i.e., 0.19 mM (acetate) vs. 0.15 mM (CK?); Fig. 4).

Removal of soluble Cr(VI) by immobilized strain Q5-1 cells

The reduction efficiencies of nine immobilization matrix combinations (from a to i) are shown in Table 2. Among the matrices for whole cell immobilization of Q5-1, the compounding parameter of ‘‘c’’ containing 4.0% PVA, 3.0% sodium alginate, 1.5% active carbon, and 3.0% diatomite showed the most effective result, which reduced 0.5 mM Cr(VI) to 0.01 mM in 17 h (97.5% removed). This Cr(VI) removal efficiency is comparable with the free Q5-1 cells (Fig. 5). By treatment of Q5-1 immobilized particles, the final concentration of Cr(VI) reached the national discharge standard level (0.5 mg l-1 & 0.01 mM; EPD 2008). Other immobilizing matrix combinations had lower Cr(VI) reduction efficiencies (Table 2). Furthermore, after incubation for 24 h, the compounding beads of ‘‘a’’, ‘‘e’’ and ‘‘h’’ (Table 2) were unstable and broken. The cells leached out slightly due to the loss of bead integrity. In contrast, the compounding beads of ‘‘c’’ showed the best characteristics of Cr(VI) removal efficiency, bead integrity and mechanical strength (data not shown).

Fig. 3 Cr(VI) reduction tests of Intrasporangium sp. Q5-1. a The growth (j) and Cr(VI) reduction (u) curves in LB medium (pH 8.0) Discussion amended with 1 mM K2CrO4;(m) LB medium (pH 8.0) amended with 1 mM K2CrO4 without bacterial inoculation. b Cr(VI) reduction of Intrasporangium sp. Q5-1 in LB medium (pH 8.0) amended with Microorganisms with the abilities of tolerance and reduction 2mM(m), 3 mM (9), 4 mM (u) and 5 mM (e) Cr(VI), respec- of Cr(VI) may be applied for environmental Cr(VI) detox- tively. c Cr(VI) reduction using repeated spiking of 1 mM K2CrO4 in ification and bioremediation (Camargo et al. 2003a). In our LB medium (pH 8.0). Error bars represent standard deviation of study, we isolated and identified a bacterium Intrasporan- triplicate tests gium sp. Q5-1 from a manganese/chromium mine soil. Q5-1 removed Cr(VI) efficiently under an aerobic condition and is Cr(VI) reduction by resting cells of strain Q5-1 highly resistant to Cr(VI). After Cr(VI) reduction, the cell and detection of the effects of different carbon shapes of Q5-1 became longer and more irregular, possibly sources on bacterial Cr(VI) reduction due to the accumulation of Cr(VI) reducing products that located inside the cells or on the cell surfaces. The reduction of Cr(VI) using washed resting cells of Q5-1 The Intrasporangium is the typical genus of the family was also tested with and without additional carbon sources Intrasporangiaceae. Reports about this family members (Fig. 4). Acetate appears to be the most effective carbon were very limited so far. I. calvum was the first published source for stimulating Cr(VI) reduction. Lactose, sucrose, species of Intrasporangiaceae (Kalakoutskii et al. 1967; and glucose slightly stimulated the Cr(VI) reduction, while Kampfer et al. 2006). To our knowledge, no strains of 123 World J Microbiol Biotechnol

0.2

0.16

0.12

0.08

0.04 ) reduction) removal / (mM) 0

Cr( - l K + e H o C CK cose an actose NAD hanol L Acetat Et Sucrose Glu Meth Carbon sources

Fig. 4 Effects of seven carbon sources (lactose–ethanol) on Cr(VI) killed bacterial cells (100°C, 10 min) that used as a negative control; reduction/removal rates. Resting cell tests of strain Q5-1 were CK? shows the resting cell tests of strain Q5-1 that previously grew in performed after incubation for 6 h at 37°C with 160 rev. min-1 LB medium without adding a carbon source. Error bars represent shaking, as described in ‘‘Materials and methods’’. CK - is the heat standard deviation of triplicate tests

Table 2 Cr(VI) removal using strain Q5-1 embedded with different immobilizing matrix combinations Immobilizing matrix combination (weight %) Cr(VI) concentration (mM) Cr(VI) removal (%) PVA Sodium alginate Active carbon Diatomite Initial (0 h) Final (17 h) a 4.0 2.0 0 1.0 0.482 ± 0.011a 0.137 ± 0.005 71.7 b 4.0 2.5 1.0 2.0 0.476 ± 0.008 0.048 ± 0.002 90.0 c 4.0 3.0 1.5 3.0 0.488 ± 0.014 0.012 ± 0.001 97.5 d 6.0 2.0 1.0 3.0 0.479 ± 0.025 0.048 ± 0.001 89.9 e 6.0 2.5 1.5 1.0 0.481 ± 0.019 0.065 ± 0.004 86.4 f 6.0 3.0 0 2.0 0.488 ± 0.006 0.115 ± 0.005 76.5 g 8.0 2.0 1.5 2.0 0.479 ± 0.021 0.050 ± 0.003 89.5 h 8.0 2.5 0 3.0 0.487 ± 0.018 0.148 ± 0.004 69.6 i 8.0 3.0 1.0 1.0 0.488 ± 0.009 0.054 ± 0.003 89.1 a Standard deviations of three independent experiments

Blank Immobilized cells Free cells Strain Q5-1 has the ability to reduce Cr(VI) either in 0.5 growing cells or resting cells (non growth). When using 0.4 resting cells for Cr(VI) reduction,the Cr(VI) was added in the resting cell resuspension solution, and it is not neces- 0.3 sary to add Cr(VI) in the culture medium before harvest of 0.2 the resting cells. A similar result has been reported by Opperman and van Heerden (2007), which suggested that a ) concentration (mM) 0.1 ‘‘chromate reductase’’ may either be constitutively

Cr( 0 expressed or the reaction was catalyzed by an enzyme with 0 3 6 9 12 15 18 other primary physiological function. Incubation time (h) In Cr(VI) reduction assay using Q5-1 resting cells, Fig. 5 Chromate reduction by free and immobilized cells of strain acetate is the most effective carbon source for stimulating Q5-1 in LB medium (pH 8.0) amended with 0.5 mM K2CrO4. Cr(VI) reduction. In two Bacillus species G1DM20 and Reduction of 0.5 mM Cr(VI) were performed using: (m) free cells; G1DM64, adding acetate increased the reduction of Cr(VI) j ( ) immobilized Q5-1cells embedded with 4% PVA, 3% alginate, (Desai et al. 2008). In other reports, NADH was very 1.5% active carbon and 3% diatomite beads, and (u) the compounding beads of 4% PVA, 3% alginate, 1.5% active carbon and 3% effective for stimulating Cr(VI) reduction (Horitsu et al. diatomite without embedment of Q5-1 cells 1983; Camargo et al. 2003b; Sultan and Hasnain 2007; Desai et al. 2008). However, in this study with Q5-1, Intrasporangiaceae have been reported to have the ability NADH showed no stimulation for Cr(VI) reduction, pos- of resistance and reduction of Cr(VI). Thus we consider sibly because that the ‘‘Cr(VI) reductase’’ of Q5-1 is not a Q5-1 as a novel Cr(VI)-reducing bacterium. NADH dependent enzyme. In addition, we found that 123 World J Microbiol Biotechnol

1 mM Cu(II) stimulated Cr(VI) reduction (data not shown), References indicating that the Cr(VI) reductase of Q5-1 may need Cu(II) as an electron-transport protector or as a co-factor in Ackerley DF, Barak Y, Lynch SV, Curtin J, Matin A (2006) Effect of electron redox center (Camargo et al. 2003b). Comparing chromate stress on Escherichia coli K-12. J Bacteriol 188:3371– 3381. doi:10.1128/JB.188.9.3371-3381.2006 with NADH, acetate is cheaper, which is a more useful APHA (American Public Health Association), AWWA (American stimulator for Cr(VI) bioremediation applications. Water Works Association), WEF (Water Environment Federa- In bacterial immobilization study, Q5-1 cells were suc- tion) (1995) Standard methods for the examination of water and cessfully embedded in the beads and exhibited a significant wastewater, 19th edn. American Public Health Association, Washington, DC chromate reducing activity. Among the matrices for whole Bae WC, Lee HK, Choe YC, Jahng DJ, Lee SH, Kim SJ (2004) cell immobilization of Q5-1, the compounding beads of 4% Purification and characterization of NADPH dependent Cr(VI) PVA, 3% sodium alginate, 1.5% active carbon and 3% reductase from Escherichia coli ATCC 33456. J Microbiol diatomite proved to be the most effective combination. 43:21–27 Bailey RW, Scott EG (1966) Diagnostic microbiology, 2nd edn. The Natural matrices have the disadvantages of abrasion and C.V. Mosby Company publisher, Saint Louis biodegradable. Synthetic matrices can conquer this disad- Camargo FAO, Bento FM, Okeke BC, Frankenberger WT (2003a) vantage with an appropriate combination (Poopal and Lax- Chromate reduction by chromium-resistant bacteria isolated man 2008). PVA is a promising type of synthetic polymer from soils contaminated with dichromate. J Environ Qual 32:1228–1233 and is not toxic to microorganisms, it is therefore very Camargo FAO, Okeke BC, Bento FM, Frankenberger WT (2003b) In suitable for entrapment of microbial cells in its polymeric vitro reduction of hexavalent chromium by a cell-free extract of 2? matrixes. In our study, using a matrix combination of nature Bacillus sp. ES 29 stimulated by Cu . Appl Microbiol and synthetic materials, the immobilized bacterium Biotechnol 62:569–573. doi:10.1007/s00253-003-1291-x Campos J, Martinez Pacheco M, Cervantes C (1995) Hexavalent remained stable and showed a satisfied Cr(VI) removal chromium reduction by a chromate-resistant Bacillus sp. strain. efficiency, indicating that the beads had stable structures and Antonie Van Leeuwenhoek 68:203–208. doi:10.1007/BF00871 the bioreaction was successfully performed inside. 816 The immobilized cells of strain Q5-1 showed a similar Cervantes C, Campos GJ (2007) Reduction and efflux of chromate by bacteria. 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