Reduction of Toxic Hexavalent Chromium by E. coli

Reduction of toxic hexavalent was studied by using four resistant strains of E. coli ASU 3, 7, 8 and 18 isolated from wastewater of EL-Malah canal located in Assuit city, Egypt. They showed relatively high minimal inhibitory concentrations (MIC) and found to be plasmid mediated with 65 and 27 Kb. E. coli ASU 7 represents the best with high resistance and reducing power of Cr (VI), so it may be a suitable candidate for bioremediation. Alternation of protein profile in SDS-PAGE of the above strain was carried out under different concentrations of chromium stress. Kew words: Chromate reduction E. coli Plasmid isolation and SDS-PAGE INTRODUCTION to high levels while others are sensitive [9]. The bacterial Heavy metals found in wastewaters are harmful but it can also be coupled to chromosomal DNA to the environment and their effects on biological [10-11]. systems are very severe. Chromium is one of the most Microorganisms can play an important role in the widely used metals in industry, such as steel production, removal of hexavalent chromium from the polluted alloy preparation, wood preservation, leather tanning, sites [12]. A wide variety of bacteria have been reported metal corrosion inhibition, paints pigments, metal plating, to reduce hexavalent to trivalent chromium under aerobic tanning, electroplating, steel manufacture and other and anaerobic conditions [13-17]. Biotransformation industrial applications [1-3]. Chromium (Cr) is an essential of chromate by chromium resistant bacteria (CRB) trace element for all living organisms. Its valence state offers an economical as well as eco-friendly option for ranged from -2 to +6. Trivalent chromium is necessary chromate detoxification and bioremediation [18]. The for fat and glucose metabolism and proper functioning main advantages of using bacterial Cr(VI) reduction of insulin [4]. Hexavalent chromium is easily soluble and are that it does not require high energy input nor toxic 100-fold more toxic than trivalent one. chemical reagents and the possibility of using Hexavalent chromium it has been recognized as one native and non-hazardous strains [19]. of the most dangerous environmental pollutants due to The objective of the present work is to characterize its ability to cause mutations, irritation, corrosion of the and evaluate plasmid mediated chromate resistant E. coli skin and respiratory tract to most microorganisms; it with high potentialities to reduce hexvalent chromium to also causes lung carcinoma in humans [5-7]. Efficient and trivalent one and analysis of their alternation of protein cheap treatments for heavy metal removal and reusing profile by SDS-PAGE under chromium stress. of spent metals from wastewater need to be developed, microbe based technologies can provide an alternative MATERIALS AND METHODS to conventional methods for metal removal [8]. The reduction of Cr (VI) to Cr (III) is therefore an attractive Determination Physicochemical Properties of Water and useful process for remediation of Cr (VI) pollution; Samples: Different polluted water samples were so many technologies have accordingly received much collected in sterile glass bottles from different sites in more concerns [5]. El-Mlalah Canal, Assiut City (Egypt), it is far about 3 km The presence of Cr (VI) in the environment plays from the main River Nile in the west site of Assiut a selective pressure on microflora and possess resistance City and exposed to domestic sewage disposal & chromate resistance is generally combined to plasmids Global J. Biotech. & Biochem., 4 (2): 98-103, 2009 99 industrial effluent. Temp, pH and Electrical conductivity Plasmid Transformation: To confirm the plasmidwere determined by water checker model Horiba U-10. encoded resistance to chromium studied, competent Heavy metals were determined according to Elith and cells of E. coli DH5 , sensitive to hexavalent chromium Garwood [20]. were transformed with respective plasmids using the Isolation of E. coli: E. coli was enumerated from of transformed E. coli DH5 were plated on tris agar wastewater samples by Most Probable Number (MPN) techniques using lactose broth medium [21]. Positive presumptive tubes show acid and gas when incubated at 37 °C for 48 hours. Biochemical Identification: Various biochemical tests (Indole, Methyl test, Voges-Proskauer, Citrate, Hydrogen sulfide on TSI, Urease, Motility, Gelatin liquefaction at 22°C, KCN, Lactose, nitrate reduction and Oxidase test) were done according to Bergey's Manual of Systematic Bacteriology [22]. Evaluation of Chromium Resistance for E. coli Strains: Minimal Inhibitory Concentration (MIC) for hexaand trivalent chromium were registered on agar plates of tris minimal agar medium using agar dilution methods and were confirmed in broth medium, for broth medium a 5 ml of tris minimal medium containing different concentrations of CrCl .6H O (0-300 ppm) and K CrO (03 2 2 4 25 ppm) inoculated with 200 μl of an 18 h old culture of the studied bacterial E. coli strain at 37°C for 2 days. The lowest concentration of heavy metals that completely preventing growth known as MIC [23]. Chromate Reduction in Liquid Medium: The chromate reduction capability of isolates was investigated under aerobic conditions in tris minimal broth medium amended with various concentrations of chromium ranged from 1 to 10 ppm at pH 7.0 [24]. Chromate reduction was suggested by Bopp and Ehrlich [25]. Broth medium inoculated with 200 μL of preculture with shaking at 150 rpm and 37°C for 48 h. Cr added after 3hr of incubation and 6+ measured at different times intervals (3-48 h). Cells were centrifuged at 10.000 xg for 10 min then the supernatant was filtrated through filter papers (Whatman no 1) then chromate reduction was quantified by measuring the decrease in absorbance at 382 nm using a Cecil spectrophotometer. Plasmid Isolation: Plasmid isolation of bacterial cells was carried out according to Birnboim and Doly [26] using alkaline lysis procedure, plasmid DNA size was The MIC of selected strains of E. coli ASU 3, 7, 8 and determined by the LabImage 1D (2006) software program. 18 were registered on tris minimal medium and confirmed standard chemical method [27]. The suspensions (100 μl) minimal media supplemented with 20 ppm of hexavalent chromium. SDS-PAGE of Proteins: The protein patterns were analyzed using SDS-PAGE according to Laemmli [28] in the first dimension. Gels were stained overnight in 200 ml of Commassie brilliant blue R-250 solution. Destaining of protein was performed in 200 ml of destaining solution which composed of 250 ml methanol, 50 ml glacial acetic acid and 200 ml distilled water with gentle shaking. Protein Analysis: The LabImage 1D (2006) program was used in molecular weight determination of proteins. RESULTS AND DISCUSSION Physicochemical Properties of Samples: The temperature, pH and Electrical conductivity corresponded to 23.7-27 °C; 7.9-8.12 and 0.34-0.35 ms/Cm, respectively. The concentration of chromium was expressed in mg/l and ranged between 0.38-0.88 mg/l. The results obtained exceeded the safe limit of WHO which is 0.05 mg/l of chromium [29]. Isolation of E. coli Isolates: E. coli was enumerated from wastewater samples by MPN techniques using lactose broth medium. Positive presumptive tubes were confirmed on Eosin Methlyene Blue (E.M.B) agar medium. Metallic green isolates were selected randomly, purified and preserved on nutrient agar for further studies. Biochemical Identification: Bacteria are gram negative rods. Various biochemical characteristics of the strains were shown in Table 1. Based on the biochemical analysis, bacteria have been identified as E. coli according to Bergey's Manual of Systematic Bacteriology [22]. Evaluation of Chromium Resistance for E. coli Strains: