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 un- der 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.