Leucobacter Chromiireducens CRB2, a New Strain with High Cr(VI) Reduction Potential Isolated from Tannery-Contaminated Soil (Fez, Morocco)

Ann Microbiol (2016) 66:425–436 DOI 10.1007/s13213-015-1125-y

ORIGINAL ARTICLE

Leucobacter chromiireducens CRB2, a new strain with high Cr(VI) reduction potential isolated from tannery-contaminated soil (Fez, Morocco)

Nezha Tahri Joutey1 & Wifak Bahafid1 & Hanane Sayel1 & Soumiya Nassef1 & Naïma El Ghachtouli1

Received: 9 March 2015 /Accepted: 25 June 2015 /Published online: 17 July 2015 # Springer-Verlag Berlin Heidelberg and the University of Milan 2015

Abstract A new chromate-reducing bacterial strain was iso- Keywords Cr(VI) reduction . Immobilization . Leucobacter lated from soil contaminated with tannery waste. Based on chromiireducens . Soluble fraction 16S rRNA gene sequence analyses, this strain was identified as Leucobacter chromiireducens CRB2. This bacterium had high multiresistance against heavy metals with a MIC of Introduction 700 mg/L Cr(VI) and was able to reduce Cr(VI) both aerobi- cally and anaerobically. The optimum pH and temperature for Chromium (Cr) is one of the most toxic heavy metals Cr(VI) reduction were pH 8.0 and 30 °C, respectively. Glyc- discharged into the environment through various industrial erol (10 mM) was the most efficient carbon source for Cr(VI) wastewater, especially from tanneries (Mythili and reduction by the strain followed by glucose. Moreover, Cr(VI) Karthikeyan 2011). Therefore, worldwide, huge amounts of reduction by L. chromiireducens CRB2 was unaltered in the chromium are dumped into the environment without any treat- presence of other oxyanions. Bacterial cells immobilized in ment, and this has become a serious health problem. Chromi- Na-alginate beads showed a high Cr(VI) reduction rates and um exists in several oxidation states, but the most stable are exhibited an ability to repeatedly reduce Cr(VI). Therefore, trivalent Cr(III) and hexavalent Cr(VI) species, with different immobilized cells were more effective than free cells in Cr(VI) chemical characteristics and biological effects (Cervantes et al. reduction. Resting and permeabilized cell assays provided the 2001). better evidence of the presence of an enzymatic chromate Conventionally, Cr(VI) is treated by physico-chemical reduction in L. chromiireducens CRB2. Assays conducted methods. However, most of these technologies need high en- with cytosolic and particulate fractions of L. chromiireducens ergy or large input of chemical reagents that may cause sec- confirmed the role of cytosolic proteins in Cr(VI) reduction. ondary environmental pollution (Komori et al. 1990). Recent- Cr(VI) reduction by L. chromiireducens was mediated by a ly, increasing attention has been paid to the use of bioremedi- soluble enzyme contained in the cytoplasm after its adsorption ation approaches through selective microorganisms capable of on the cell surface. To the best of our knowledge, this is the reducing Cr(VI) to the less toxic and insoluble Cr(III) (Tahri first report studying parameters affecting Cr(VI) reduction and Joutey et al. 2013). describing Cr(VI) removal mechanism by strain of Microbial Cr(VI) reduction was first reported in the late L. chromiireducens. 1970s when Romanenko and Koren’kov (1977) observed Cr(VI) reduction capability in Pseudomonas spp. grown under anaerobic conditions. Since then, several researchers have isolated new microorganisms that catalyse Cr(VI) reduction * Naïma El Ghachtouli under varying conditions (Viti et al. 2003). Other researchers [email protected] have also used consortium cultures for Cr(VI) remediation (Cheung and Gu 2003; Tahri Joutey et al. 2011). Leucobacter 1 Microbial Biotechnology Laboratory, Faculty of Sciences and sp. belongs to metal stressed communities, and a few chro- Techniques, Sidi Mohamed Ben Abdellah University, Route mate tolerant strains of this genus have been reported from Immouzer, P. O. Box 2202, Fez, Morocco activated sludge and sediments subjected to chromium 426 Ann Microbiol (2016) 66:425–436 contamination (Sarangi and Krishnan 2008). The original de- DNA was extracted using thermal shock protocol: an iso- scription of Leucobacter chromiireducens as a novel species lated colony from a young LB-agar culture of the isolate was of the genus Leucobacter and the validation of the species mixedwith50μL of sterile distilled water in 1.5 mL name have been reported (Morais et al. 2004, 2005). Howev- microcentrifuge tube. The tube was frozen at −20 °C for er, to our knowledge, no study on the mechanism and the 30 min and then heated at 95 °C for 3 min. This thermolysis parameters affecting Cr(VI) reduction by L. chromiireducens procedure was repeated twice. After centrifugation at 7000 g have been conducted. The physiological mechanisms in- for 10 min, 2 μL of the supernatant was used in the amplifi- volved in chromate reduction vary widely among species. In cation reaction. The reaction mix was prepared in a final some cases, the enzyme-catalyzed reaction is membrane asso- reaction volume of 20 μL and contained 4 μL of Taq buffer ciated, whereas in others the enzyme is in the soluble form (5x), 1.2 μLofMgCl2 (25 mM), 4 μL of dNTPs (1 mM), either extracellular or cytosolic (Tahri Joutey et al. 2013). 2 μL of fD1 (10 μM), 2 μLofRS16(10μM), 0.2 μLof

Methods using free cells for remediation suffer due to Taqpolymerase(5U/μL), 4.6 μLofpureH2O, and 2 μLof Cr(VI) toxicity and cell damage. Whole cell immobilization the DNA. The amplification protocol was carried out in a has advantages over free cells in that they are more stable, Techgene thermal cycler under the following conditions: de- thier ease of regeneration, possibility of reuse, easier solid– naturation at 94 °C for 5 min, 35 cycles of denaturation at liquid separation, minimal clogging in continuous systems, 94 °C for 30 s, primers annealing at 55 °C for 45 s, and and is attracting attention worldwide (Poopal and Laxman primer extention at 72 °C for 1 min 30 s; final extension was 2008). Immobilization of bacteria for Cr(VI) reduction has performed at 72 °C for 10 min. For each reaction, a negative been reported in continuous culture by immobilized cells control (without template DNA), and a positive control was of Microbacterium liquefaciens MP30 (Pattanapipitpaisal included. Efficient amplification was confirmed by gel elec- et al. 2001), Bacillus sp. ES 29 (Camargo et al. 2004), trophoresis on 1 % agarose gel and visualized by ethidium Serratia marcescens as a stable biofilm (Mondaca et al. bromide. DNA sequencing was performed using an ABI 2002), and Pseudomonas immobilized in agar–agar films 3130 (Applied Biosystems) according to the manufacturer’s on the cellulose acetate membrane (Konovalova et al. instructions. 2003). The sequence was initially analyzed at the National Center The present work is aimed at isolating and characterizing of for Biotechnology Information (NCBI) server (http://www. anovelL. chromiireduscens strain able to reduce Cr(VI). This ncbi.nlm.nih.gov) using the BLAST (blastn) tool to identify is also the first report trying to elucidate the mechanisms of and download the nearest neighbor sequences from the NCBI Cr(VI) removal by this bacterium. database. A part of the 16S rRNA (487 bp) was submitted to the EMBL Nucleotide Sequence Database (also known as EMBL-Bank) with accession no. HE963772. Materials and methods All the sequences were aligned using the Clustal W 1.6 program at (http://www.ebi.ac.uk/clustalw). The phylogenetic Isolation, characterization, and identification tree was constructed using aligned sequences by the neighbor of the bacterial strain joining (NJ) algorithm using Kimura 2 parameter with more than 1000 replicates in Molecular Evolutionary Genetics Anal- The strain CRB2 used in this study was isolated from a ysis (MEGA version 5) software (Tamura et al. 2011). chromium-contaminated site located in the region of Fez (Morocco). Growth was carried out in sterile Luria Broth me- Evaluation of heavy metals tolerance dium (LB) consisting of peptone 10 g/L, NaCl 10 g/L, and yeast extract 5 g/L in 1000 mL distilled water. Cell morphol- Tolerance of the strain CRB2 against increasing concentra- ogy was examined after Gram staining, as described by Bailey tions (50–1500 mg/L) of Cr(VI), Cr(III), Cu(II), Zn(II), Ni(II), and Scott (Bailey and Scott 1966). Biochemical analyses were Mn(II), and Co(II) on LB-agar plates was evaluated until the performed according to the Bergey’s manual (Holt et al. strain was unable to produce colonies on the agar plates. 1994). Based on this evaluation, minimum inhibitory concentration Molecular identification approach involved the use of 16S (MIC) was determined after 48 h of incubation at 30 °C. rDNA analysis; this methodology, according to Strous et al. Heavy metals were filter sterilized and added separately to (1999), allows the identification of various bacteria from the the LB-agar medium. environment. The 16S rRNA gene fragment of the isolate was amplified by PCR (polymerase chain reaction). The rDNA Cr(VI) reduction by growing cells of the isolate 16S regions were amplified using primers fD1 (5′AGAGTT TGATCCTGGCTCAG3′) and RS16 (5′TACGGCTACCTT Chromium reduction experiments were realized in 150 mL GTTACGACTT3′) (Weisberg et al. 1991). Erlenmeyer flasks containing 50 mL of culture media (LB or Ann Microbiol (2016) 66:425–436 427

M63 medium). The medium with appropriate pH (adjusted strength, rigidity, and porous characteristics to the biological with 0.1 M HCl or NaOH) was supplemented with the desired material.

Cr(VI) concentration, inoculated with an overnight bacterial The variation of the concentration of CaCl2 solution and culture, and incubated in an incubator shaker at the appropri- time of the reaction leads to the formation of alginate ate temperature with shaking (150 rpm). microbeads with various degree of cross linking (Nita et al. For Cr(VI) reduction studies, solution of potassium dichro- 2007). In this study, to carry out immobilization, 0.1 M of mate as source of Cr(VI) was filter sterilized and then added to CaCl2 solution was prepared, sterilized and kept at 4 °C for the medium aseptically. chilling. According to Trevors et al. (1992) low concentrations

Effect of oxic and anoxic conditions on Cr(VI) reduction of CaCl2 (0.05 to 0.1 M) and longer polymerisation time are by the bacterial strain were studied. Since Cr(VI) reduction preferable to have more homogeneous and more resistant was better in oxic conditions, Cr(VI) reduction experiments beads. The next step was to dissolve sodium alginate (effect of pH, temperature, electron donors, and ions) were (1.5 %) in hot distilled water with constant stirring on mag- performed in oxic conditions. To set anaerobic growth condi- netic stirrer and to autoclave this solution only 15 min at tions, flasks were incubated in anaerobic jar (Don Whitley 121 °C to make stable beads. After cooling, cell suspension Scientific LTD) using tablet of sodium bicarbonate and acti- previously prepared was added and stirred on magnetic stirrer vator solution. aseptically. The alginate biomass slurry was introduced into

To study the optimum pH condition, LB medium contain- 0.1 M CaCl2 for polymerization and bead formation using ing 100 mg/L of Cr(VI) with different initial pH (from 4.0 to 5 mL sterile syringe. The resultant beads were of about

9.0) was inoculated with 1 % of the cell culture (OD600=1.0) 3 mm diameter (beads may be stored at 4 °C in CaCl2 solution and incubated at 30 °C with 150 rpm shaking up to 2 days. until use). After gelling, the microbeads were placed in sterile The effect of temperature was tested in the optimum pH at distilled water to remove unreacted material and low molecu- temperatures 25, 30, 35, 4,0 and 45 °C as described above. lar weight by products. Beads were washed twice with sterile The effect of different electron donors on Cr(VI) reduction distilled water and added aseptically to 50 mL LB medium by the isolate was tested in M63 medium, commonly used in containing 100 mg/L Cr(VI) in a 250-mL flask. The flasks microbiology (KH2PO4 13.6 g/L, KOH 8.3 mL (130 g/L solu- were incubated at 30 °C with 150 rpm shaking. Samples were tion), (NH4)2SO4 10 mL (20 % solution), (FeSO4·7H2O) 1 mL taken at regular intervals and the Cr(VI) concentration was (0.1 % solution), thiamine 1 mL (0.05 %), (MgSO4·7H2O) detected as described below. For free cells, cell suspension 1 mL (20 % solution)) (Adeniji 2004), adjusted to pH 8 with was used to inoculate LB medium amended with 100 mg/L NaOH (0.1 M) and supplemented with 10 mM of glucose, Cr(VI). Controls are prepared by following the same steps, fructose, glycerol, acetate, citrate, NADPH, tryptophan, and except that instead of adding the cellular suspension sterile benzoic acid as a unique electron donor, and was amended with distilled water was added. 100 mg/L Cr(VI). The continuous reduction in non modified LB medium of The effect of some ions was studied in LB medium repeated Cr(VI) inputs of 100 mg/L by free and immobilized amendedwith10mMofnitrate(NH4NO3), sulphate cells was also studied. To study Cr(VI) reduction by free and (Na2SO4) and carbonate (Na2CO3). Cr(VI) was added at immobilized cells in a modified medium, after each cycle 100 mg/L. Cultures were incubated in a shaker at 30 °C and (48 h incubation) cells and beads were washed twice with 150 rpm. sterile distilled water and were resuspended in fresh LB medium amended with the same Cr(VI) concentration. Bacterial immobilization Localization of chromate-reducing activity Preparation of the bacterial culture Resting cell assay One hundred milliliters of sterile LB medium was inoculated with the strain CRB2. After 24 h incubation at 30 °C and Cells of L. chromiireducens were grown overnight in 50 mL under 150 rpm shaking, cells recorded by centrifugation LB medium and harvested by centrifugation at 7000 g at (7000 g, 10 min) were washed twice with sterile distilled 4 °C. Cell pellet was washed twice with 0.05 M potassium water and suspended in 15 mL of sterile distilled water. This phosphate buffer, pH 7 and suspended in the same buffer. cell suspension serves also as free cells. These cell suspensions were spiked with 25 and 50 mg/L Cr(VI). The tubes were vortexed briefly for 1–2 min and Preparation of alginate beads incubated under shaking at 30 °C. Heat killed cells were used as controls. At the end of incubation, tubes were centrifuged Immobilization of the biomass with in a suitable matrix pro- and remaining Cr(VI) was estimated from the supernatant vide a physical support for cells, ideal size, mechanical (Soni et al. 2012). 428 Ann Microbiol (2016) 66:425–436

Permeabilized cell assay Statistical analysis

Overnight grown cells in 200 mL LB medium were harvested Observations were made and all the experiments run in tripli- by centrifugation (6000 g for 20 min at 4 °C), washed twice cate. Each time three readings were taken. The data obtained with 20 mL of 10 mM Tris–HCl buffer pH 7.2 and suspended was statistically analyzed with Student's t-test and analysis of in 20 mL of the same buffer. Toluene (0.1 mL) and Triton X variance (ANOVA) to determine significance of differences 100 (0.2 %, v/v) were added to 9.9 mL of the cell suspension between means. The differences were statistically significant and vortexed to permeabilize the cells (Thacker et al. 2006). when the p-value was less than 0.05. To find out which means After adding chromium to a final concentration of 25 mg/L the are significantly different from others, Fisher's Least Signifi- permeabilized cells were incubated at 30 °C under shaking for cant Difference (LSD) procedure was done (with 95.0 % con- 6 h. Permeabilized cells, which were heated at 100 °C for fidence level). R, Version 2.15.2 using the package (agricolae) 30 min, were used as control. After incubation, the perme- was used as statistical analysis software. abilized cells were centrifuged (6000 g for 20 min at 4 °C) and Cr(VI) concentration was determined from the supernatant (Thacker et al. 2006). All assays were done in triplicate. Results and discussion The evaluation of cells permeabilization was done by spreeding the cell suspension on LB agar plates. After 24 h Bacterial identification and phylogenetic analysis incubation at 30 °C, no visible colonies were observed. Colonies of the isolate CRB2 are circular, entire, low convex, Cell fractionation smooth, opaque, and cream-colored. It forms Gram positive, irregular, rod-shaped cells. The isolate was identified using Bacterial cultures grown [with and without Cr(VI)] overnight molecular identification. The molecular definition of genus in 200 mL LB medium, were harvested by centrifugation at states that 16S rRNA sequence similarity should be superior 6000 g for 20 min at 4 °C, washed twice with 20 mL of 10 mM or equal to 97 %. Similarity superior to 99 % indicates iden- Tris HCl buffer pH 7.2 and were suspended in 30 mL of the tical species (Drancourt et al. 2000). The sequences were com- same buffer. Cells were disrupted by sonication for 5 or pared to the EMBL, GenBank, DDJB, and PDB databases, 10 min (Elma X-TRA 50H) in cold conditions (Thacker et al. using the BLAST NR 2.2.11 program through the National 2007). The resultant homogenate was centrifuged at 6000 g Center for Biotechnology Information (NCBI) and revealed a for 30 min at 4 °C. Supernatant obtained after harvesting of similarity of 99 % with L. chromiireducens. This is the first the cells was filter sterilized and used for chromate reductase isolate obtained and characterized in Morocco. The phyloge- assay. Chromium was added to final concentrations of 25 and netic tree derived from 16S rRNA gene sequence of 50 mg/L. The pellet consisting of particulate fraction was L. chromiireducens CRB2 HE963772 and sequences of clos- suspended in 30 mL of 10 mM Tris–HCl buffer pH 7.2 and est phylogenetic neighbors obtained by NCBI BLASTn anal- assayed for Cr(VI) reduction. Crude extract and pellet heated ysis shown in Fig. 1 demonstrates the relationships among at 100 °C for 30 min served as control. All the flasks were selected isolates. The NJ-tree was constructed using the incubated at 30 °C for 24 h with shaking. Aliquots of sample neighbor-joining algorithm with Kimura 2 parameter dis- were withdrawn and analyzed for Cr(VI). tances in MEGA 5.1 Beta4 software (Tamura et al. 2011). Furthermore, the isolate is oxidase negative and catalase Analytical methods positive. Nitrate is not reduced; arginine dihydrolase and ure- ase are not produced. Esculine and gelatin are not hydrolized. Assessment of Cr(VI) reduction by the bacterial isolate was Glucose is fermented. L-Rhamnose, D-galactose, D-sorbitol, carried out by withdrawn aliquots of cultures, after centri- D-cellobiose, D-raffinose are assimilated. These characteristics fugation (10 min at 7000 g), chromium reducing activity appear also in the description of the isolate L. chromiireducens was estimated as the decrease in chromium concentration in sp. nov. (Morais et al. 2004), the first and only isolate reported supernatant with time using Cr(VI) specific colorimetric to have tolerance and reducing ability of Cr(VI) as indicated by reagent S-diphenyl carbazide (DPC) 0.25 % (w/v) prepared Scopus analyze result using L. chromiireducens as research by acetone to minimize deterioration as described by Eaton words. et al. (1995). Spectrophotometric measurements of the pink colored complex formed from 1,5-diphenyl carbazide and Evaluation of heavy metal resistance Cr(VI) in acidic solution were made immediately at 540 nm. Isolation of bacteria from metal-polluted environment would The growth of cells was routinely monitored by measuring represent an appropriate practice to select metal resistant optical density (OD) with spectrophotometer at 600 nm. strains that could be used for heavy metal removal and Ann Microbiol (2016) 66:425–436 429

Fig. 1 Neighbor-joining tree based on 16S rRNA gene sequences of Leucobacter chromiireducens analysed by MEGA 5.1 Beta4 software (Tamura et al. 2011). Isolate strain obtained during this study is marked with asterisks. Numbers at nodes show occurrences in bootstrap sample and provide an estimate of confidence of the analysis. Accession numbers are mentioned after the name of the isolate followed by the sequences bank. Scale bar indicates 0.002 Base differences per position

bioremediation purpose (Malik 2004). Leucobacter resistance and ability to reduce this metal ion, but was not chromiireducens CRB2, isolated from soil contaminated with isolated from a chromium-enriched environment (Takeuchi waste tanneries, showed resistance against 700 mg/L of et al. 1996). Howerver, Cr(VI) resistance or reducing ability Cr(VI). The order of metals toxicity of this isolate was found of Leucobacter luti sp. nov., and L. alluvii sp. nov., isolated to be Co(II)=Cr(III)=Cu(II) (1000 mg/L)>Zn(II)=Mg(II) under chromium stress, was similar to the other strains of the (800 mg/L)>Cr(VI)=Ni(II) (700 mg/L). genus Leucobacter and was not related to the Cr(VI) concen- The mechanisms of bacterial resistance towards toxic tration of the environment they came from. This probably metals are different from one isolate to another, such as metal shows that in metal stress environments bacteria subsist in efflux channels, metal resistance plasmids, adsorption uptake, microenvironments with different concentrations of the metal DNA methylation, and metal biotransformation either directly according to their metal resistance ability (Morais et al. 2006). by specific enzymes or indirectly by cellular metabolites Members of the genus Leucobacter are known to exhibit chro- (Camargo et al. 2003). The best characterized mechanisms mate resistance. In fact, both strains L. chromiireducens sp. comprise efflux of chromate ions from the cell cytoplasm nov, and Leucobacter aridicollis sp. nov., as well as the type and reduction of Cr(VI) to Cr(III) (Ramírez-Díaz et al. strain of Leucobacter komagatae were resistant to 250 mg/L 2008). This difference gives various resistance profiles to Cr(VI) (Morais et al. 2004; Halpern et al. 2009). heavy metals. Furthermore, comparing metal resistance levels with values reported in the literature is not possible due to Growth and Cr(VI) reduction under aerobic different types of media and growth conditions employed. and anaerobic conditions Francisco et al. (2002) reported that Cr(VI) resistance and reduction are both shared abilities, probably reflecting hori- Cr(VI) reduction was studied under aerobic and anaerobic zontal genetic transfer resulting from selective pressure in en- conditions in LB medium pH 7 with an initial Cr(VI) concen- vironments contaminated with Cr(VI). However, Morais et al. tration of 100 mg/L. Results are shown in Table 1. It was (2004) reported that this characteristic does not seem to be a observed that under aerobic conditions, chromate reduction characteristic acquired by lateral gene transfer from other was carried out three times more efficiently compared with chromium-resistant organisms that colonize the environment anaerobic conditions. Furthermore, growth was reduced to from which the strains were isolated, but to be a common half following preincubation in an anaerobic jar. feature of the strains of the genus Leucobacter since the type The strain CRB2 was able to reduce Cr(VI) either aerobi- strain of L. komagatae also showed the same degree of Cr(VI) cally or anaerobically. However, as large areas have to be 430 Ann Microbiol (2016) 66:425–436

Table 1 Cr(VI) reduction and growth of L. chromiireducens CRB2 under aerobic and anaerobic conditions after 48 h incubation

Aerobic conditions Anaerobic conditions

Cr(VI) reduction (%) 82.97±2.41a 22.67±2.44b Growth: OD (600 nm) 2.342±0.268 a 1.274±0.164b

Values shown are mean±standard deviation. For the same parameter values with different letters are significantly different (p-value<<0.05) bioremediated under field condition, provision of aeration may not be economically feasible. Therefore, the anaerobic reduction bioremediation option is sometimes preferable though it is slightly less efficient than the aerobic system. It is useful to recall that aerobic reduction is thought to be a detoxification where cells use a soluble enzyme to reduce Cr(VI) to Cr(III) internal or external to the plasma membrane. 2− Reduction may also proceed through the use of CrO4 as a terminal electron acceptor during anaerobic respiration. As such, this activity is associated with the membrane where cytochromes have been implicated (Lovley and Coates 1997).

Effect of initial pH and temperature on bacterial growth and Cr(VI) reduction capacity

Figure 2a shows the influence of initial pH (4 to 9) on Cr(VI) reduction yield and growth of L. chromiireducens CRB2 after 48 h incubation. Cr(VI) removal showed a pH-dependent pro- file. Indeed, the Cr(VI) reduction by L. chromiireducens CRB2 Fig. 2 Effect of different initial pH (a) and temperature incubation (pH 8) occurred in a wide pH range from neutral to basic pH, with an (b) on Cr(VI) removal and growth of L. chromiireducens CRB2 in LB optimum at pH 8 for Cr(VI) reduction and pH 9 for growth, medium amended with 100 mg/L of Cr(VI) after 48 h incubation at 30 °C. For Cr(VI) removal, means with different letters are significantly different indicating the alkaliphilic nature of L. chromiireducens CRB2. (at 5 %, LSD-Fisher) At pH 6, just 20 % of Cr(VI) removal by L. chromiireducens CRB2 was observed. Bacterial growth and Cr(VI) reduction were completely ceased at acidic conditions (pH 4 and 5). Figure 2b shows that growth and reduction of Cr(VI) were The pH value is an important index reflecting the microbial dependent on incubation temperature. In fact, growth and activity. Various works showed that the optimal pH conditions Cr(VI) reduction were observed at 25, 30, and 35 °C. Optimal for growth and Cr(VI) reduction vary according to isolates. Cr(VI) reduction was directly related to the optimum temper- Brucella sp. could grow at pH range of 5.0–9.0, optimum pH ature for growth of the isolate, with an optimum of growth and being7(Thackeretal. 2007). For Pseudomonas fluorescens Cr(VI) reduction at 30 °C, while growth and Cr(VI) reduction and Bacillus sp., the maximal Cr(VI) reduction was observed at were strongly inhibited at elevated temperature 40 and 45 °C. pH 7.0 to 8.0 (Parameswari et al. 2009). For, Bacillus subtilis, A similar result was obtained by Sarangi and Krishnan (2008) optimum pH was 9 for Cr(VI) reduction (Mangaiyarkaras et al. for Leucobacter sp. KCH4; they reported that chromate reduc- 2011). Morais et al. (2004) studied Cr(VI) reduction in PY-BHI tase showed optimum activity at 30 °C and lost activity medium adjusted to pH 7.0 and reported that strains within the completely at 40 °C. It should be noticed that the optimal genus Leucobacter reduced 50 mg/L Cr(VI). But they did not Cr(VI) reduction depend mostly on the optimum growth study optimal conditions for Cr(VI) reduction by these strains. temperature. Enterococcus gallinarum (Sayel et al. 2012)andBacillus sp. Zhang and Li (2011) have reported that, since Cr(VI) re- KSUCr5 (Abdelnasser et al. 2011) reduced Cr(VI) in an opti- duction is enzyme-mediated, temperature and pH changes mum pH of 10. Cr(VI) reduction at high pH conditions is im- may affect the enzyme ionization rate and the protein folding, portant for many bioremediation efforts as many toxic metals consequently affect enzyme activity. containing industrial effluents and contaminated soils are in Many bacterial strains have been shown to mediate reduc- alkaline pH (Stewart et al. 2007). tion of Cr(VI) to Cr(III). Table 2 shows a comparison of Ann Microbiol (2016) 66:425–436 431 results obtained with CRB2 at pH 8 and 30 °C, with literature Table 3 Effect of electron donors on Cr(VI) reduction by data indicating Cr(VI) reduction at various conditions tested. L. chromiireducens CRB2 in M63 medium (pH8) These results clearly indicate the high potential of Electron donor Cr(VI) removal (%) Bacterial growth (OD600) L. chromiireducens CRB2 in Cr(VI) reduction in comparison c with previously isolated strains. No donor (Control) 0.0±0.00 0.0±0.00 Glycerol 47.91±2.62a 1.40±0.21 Glucose 30.33±1.84b 1.20±0,32 Effect of different carbon sources on Cr(VI) reduction Fructose 1.55±0.32c 0.60±0.00 Acetate 1.23±0.59c 0.49±0.01 Cr(VI) reduction in the minimal medium M63 amended with Citrate 1.33±0.39c 0.23±0.02 glucose and glycerol was significantly different from that in NADPH 1.88±0.48c 0.33±0.01 unamended control, which suggests that glucose and glycerol Tryptophan 1.65±0.30c 0.23±0.05 were used as the sole source of carbon and energy for chro- c mate reduction by L. chromiireducens CRB2, while Cr(VI) Benzoate 1.18±0.14 0.03±0.00 reduction by L. chromiireducens CRB2 in M63 medium Values shown are mean±standard deviation. a, b Significantly different amended with fructose, acetate, citrate, NADPH, tryptophan, from the value for the unamended control and benzoate was not significantly different from that observed without an exogenous donor (Table 3). Similarly, precipitate formation. Similar changes in color were recorded Dey and Paul (2012)havereportedthatCr(VI)by by Pal and Paul (2004). The blue precipitate formed corre- Arthrobacter sp. is accelerated by glycerol and glucose. Aer- sponds to Cr(OH)3 indicating Cr(VI) reduction to Cr(III) obic Cr(VI) reduction by Agrobacterium radiobacter, Bacillus (Zhu et al. 2008). cereus, Escherichia coli ATCC 33456, and P. flu orescens LB300 used glucose as an electron donor (Wang and Shen Effect of oxyanions on Cr(VI) reduction 1995). In addition, glycerol is a major byproduct of several by L. chromiireducens CRB2 industrial processes. Its use by L. chromiireducens as a sole 2− − − carbon and energy source for Cr(VI) reduction is significant The role of oxyanions (SO4 ,NO3 and CO3 )ascompeting for bioremediation processes. electron acceptors in Cr(VI) reduction was investigated. Sul- We note that Cr(VI) reduction in M63 medium was accom- fate is of particular importance because the ability of Cr(VI) panied by a change in yellow color of the medium and anions to overcome the permeability barrier of a prokaryotic

Table 2 Cr(VI) reduction by L. chromiireducens CRB2 in comparison with some other Cr(VI) reducing bacterial strains reported in the literature

Bacterial strain Culture conditions Initial Cr(VI) Cr(VI) reduction Reference concentration (mg/L)

Leucobacter chromiireducens L-9T PY-BHI medium adjusted 50 All the strains reduced 1.0 mM Cr(VI) to Morais et al. and Leucobacter aridicollis L-1T to pH 7.0 Cr(III). (Reduction rate is not indicated) (2004) Pseudomonas sp. strain S4 LB medium (pH 7.0) at 30 °C 160 64.4 % within 72 h Farag and Zaki with orbital shaking (2010) (200 rpm). Staphylococcus aureus and LB medium (pH 7.0) at 37 °C, 20 100 %, respectively, in 6 and 24 h Ilias et al. Pediococcus pentosaceus 150 rpm (2011) B2, B4, and B9 Belonging to the LB medium (pH 8.0), at 37 °C, 50 74.1, 73.14, and 61.5 %, after 120 h Sharma and genus Bacillus 200 rpm 100 42.15, 73.4, and 60 %, after 120 h Adholeya 200 40.75, 48.88, and 39.39 %, after 120 h (2012) Halomonas sp. M-Cr LB medium (pH 10) a 30 ° C, 50 60 % after 24 h Mabrouk et al. under shaking at 120 rpm (2014) Enterobacter sp. HT1 LB broth (pH 7.0) at 30 °C and 10 100 % within 20 h Marjangul 100 rpm. 20 100 % within 72 h et al. (2014) 30 100 % within 96 h Bacillus pumilis, Exiguobacterium, DeLeo and Ehrlich medium 200 51, 39, and 41 %, respectively, after 24 h Rehman and and Cellulosimicrobium (pH 3.0) and 37 °C 400 24, 19, and 18 %, respectively, after 24 h Faisal cellulans (2015) Leucobacter chromiireducens LB medium (pH 8.0) at 30 °C, 100 100 % within 48 h This study CRB2 150 rpm 432 Ann Microbiol (2016) 66:425–436

2 cell can be attributed to the chemical similarity between CrO4 − 2− and SO4 ions (Mabbett and Macaskie 2001). Cr(VI) reduction by L. chromiireducens CRB2 was unaltered in the − − presence of 10 mM of nitrate (NO3 ) carbonate (CO3 )and 2− sulphate (SO4 ) in comparison with unamended control (Fig. 3), indicating that Cr(VI) is a better electron acceptor than these oxyanions, as reported by Philip et al. (1998). In addition, the ability of L. chromiireducens CRB2 to withstand 2− 10 mM of SO4 indicated that Cr(VI) uptake via the sulphate uptake pathway may not be interfered by the presence of sulphate (Zainul et al. 2007).

Cr(VI) reduction by free and immobilized cells of L. chromiireducens CRB2 Fig. 4 Cr(VI) reduction by free and immobilized cells of strain CRB2 in Figure 4 shows that the reduction of 100 mg/L of Cr(VI) LB medium (pH8) amended with 100 mg/L Cr(VI) at 30 °C occurred completely after 32 h incubation with immobilized cells in comparison with 48 h by the free cells. This shows the Cr(VI) up to six cycles (48 h for each cycle) with the same efficiency of immobilized cells of strain CRB2 in Cr(VI) re- efficiency continuously in the non-modified medium without moval. Poopal and Laxman (2008) and Sikander and Shahida addition of any extra nutrients, while Cr(VI) reduction by (2007) also reported the higher Cr(VI) reduction with washed immobilized cells in modified LB medium became immobilized cells of Streptomyces griseus and Ochrobactrum lower up to the third cycle. Free cells were able the reduce intermedium strain SDCr-5 respectively compared to free 100 mg/L of Cr(VI) with the same efficiency for two cycles in cells, while immobilized cells of Intrasporangium sp. Q5-1 the non-modified medium, whereas this efficiency was main- showed a similar Cr(VI) reduction rates to that of free cells tained until the third cycle using washed cells in a fresh LB (Yang et al. 2009). medium after Cr(VI) reduction ability became lower. Similar- ly, Farag and Zaki (2010) have reported that Na-alginate Cr(VI) reduction tests using repeated addition of Cr(VI) immobilized Pseudomonas strain could be reused three times aliquots without losing their chromium reduction activity for 3 days, while Pang et al. (2011) have reported that the Cr(VI) reduc- Free cells and Na-alginate immobilized cells were tested in tion efficiency of immobilized Pseudomonas aeruginosa cells several consecutive chromium (inputs of 100 mg/L Cr(VI)) declined significantly at the seventh time. They explained this reduction experiments to investigate the possible deactivation by the loss of P. aeruginosa of reductase in the long-term of cells with repeated use in non-modified medium (Fig. 5a) shaking incubation and repeated wash, as well as the deterio- and modified medium with washed cells (Fig. 5b). It was ration or mutation of P. aeruginosa (Farag and Zaki 2010). found that the immobilized bacteria could be used to reduce The bacterial cells of Intrasporangium sp. Q5-1 were able to reduce Cr(VI) after four times repeated addition of 50 mg/L

Cr(VI) K2CrO4, after Cr(VI) reduction ability became lower (Yang et al. 2009). Thus, immobilized cells are preferable for Cr(VI) removal due to their capability to be used several times in non- modified medium without nutrients amendments with the same efficiency, making them more economical. Furthermore, immobilized cells have advantages over free cells in being more stable, easier to reuse, and with minimal clogging in continuous systems (Yang et al. 2009). In addition, immobilized bacteria cells are protected from the action of chromate at high levels (Konovalova et al. 2003).

Chromate reduction by resting and permeabilized cells

Fig. 3 Reduction of 100 mg/L Cr(VI) in the presence of 10 mM Resting cells were able to reduce only 20 % of Cr(VI) initially oxyanions by L. chromiireducens CRB2 in LB medium added to the medium, while, permeabilized cells were able to Ann Microbiol (2016) 66:425–436 433

Chromate reduction by cell extracts

Chromate reduction assays were followed using initial concentrations of 25 and 50 mg/L Cr(VI) with ultrasonicated cytosolic fraction or cell-free extract and membrane fraction (ultrasonicated pellet). As observed from Table 5,assayscon- ducted with cytosolic and particulate fraction of L. chromiireducens CRB2 confirmed the role of cytosolic proteins in Cr(VI) reduction, since low Cr(VI) reduction was obtained with the particulte fraction showing a probable Cr(VI) adsorption before its reduction. Resting and permeabilized cell assays provided the better evidence that Cr(VI) reduction occurs in cytosolic fraction as observed in previous findings of Mistry et al. (2010) and Sayel et al. (2012). Cr(VI) reduction by resting cells was carried out in potassium phosphate buffer without amendment of any electron donor. This suggests that endogenous electron donors (either through internal reserves or by endogenous decay) might be used for metal reduction (Wang and Shen 1995). Permeabilization with Tween 80 and Triton X-100 resulted in increased Cr(VI) reduction, indicating that cytoplasmic proteins were released and the strain reduced Cr(VI) through soluble cytosolic reductase and not through membrane- associated reductase. The low ability to reduce Cr(VI) by boiled cell-free extract, which served as control, showed that reduction process is enzymatic and not due to chemical reaction and may be preceded by absorption of Cr(VI) to some functional groups located on the cell surface of the bacterial strain (Tahri Joutey et al. 2013). Desai et al. (2008)suggested that a soluble chromate reductase associated with the cytoplasmic membrane catalyzed Cr(VI) reduction by Pseudomonas Fig. 5 Repeated 100 mg/L Cr(VI) reduction in non modified medium (a) sp. G1DM21 by transferring initial one electron to Cr(VI) to and modified medium (b) by free and immobilized cells of L. chromiireducens CRB2. (Cycle is 48 h incubation) form an intermediate Cr(V), followed by two electron transfer for Cr(III) formation. We can also note that there is not a significant difference reduce about 60 % of Cr(VI). We also notice that there is not a between Cr(VI) reduction by resting and permeabilized cells significant difference between Cr(VI) reduction by resting and grown with or without Cr(VI). Therefore, the presence of permeabilized cells grown in the absence or presence of Cr(VI) is not required for enzyme production. Then Cr(VI)- 10 mg/L Cr(VI) (Table 4). reducing activity of L. chromiireducens is constitutive.

Table 4 Reduction of Cr(VI) by resting and permeabilized cells at different initial Cr(VI) concentration. Cells grown in absence and in presence of Cr(VI)

Grown in absence of Cr(VI) % Cr(VI) residual Grown in presence of Cr(VI)* % Cr(VI) residual

Cr(VI) (mg/L) 25 50 25 50

Time (h) 0 6 0 6 0 6 0 6

Resting cells Control 100 88.21 100 92.90 100 89.66 100 96.77 Expt. 100 86.00 100 80.33 100 88.54 100 98.13 Permeabilized cells Control 100 96.00 100 97.33 100 90.54 100 98.13 Expt. 100 34.00 100 37.00 100 39.80 100 48.03

*10 mg/L 434 Ann Microbiol (2016) 66:425–436

Table 5 Cr(VI) residual (%) obtained by cytosolic and Sonication time: 5 min Sonication time: 15 min particulate fraction of L. chromiireducens CRB2 Initial Cr(VI) concentration (mg/L) 25 50 25 50

Time(h) 024024024024

Cytosolic fraction Control 100 98.31 100 94.96 100 99.86 100 97.65 Expt. 100 21.04 100 32.15 100 24.04 100 35.01 Particulate fraction Control 100 91.04 100 92.15 100 90.04 100 91.01 Expt. 100 91.04 100 92.15 100 90.04 100 91.01

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