Discussion The eradication of heavy metals is very necessary in order to prevent their acce ss from polluted to the terrestrial

environment. The untreated solid waste and e ffluents, comprising of higher concentration of heavy metals have posed a great risk regarding environmental pollution. According to EPA, chromium is among domi nant environmental pollutants in USA. The physical as well as chemical methods a re used for the removal of toxic chromium from the environment. But their solid waste produced is difficult to treat and dispose. So, the use of microorganisms for this purpose is more environments friendly and is an inexpensive procedure. As a result of this method, the damage to the environment is very less as compar ed to other methods, as this happens in in-situ. Heavy metals are taken up by the cells by binding to membrane components, protei ns or nucleic acids. The thick layer of peptidoglycan present in gram positive b acteria comprises of techioc acid, which acts as an important metal chelator. Wh ile the same function is performed by the outer membrane in gram negative bacter ia. The metal-protein binding occurs through sulfhydril groups (Singh and Tripat hi, 2007). The chromium reduction catalyzed y bacteria can be enhanced by optimizin g the growth conditions including temperature, pH, salt concentrations and prese nce of ions.(add any paper which has studied the ph and temp. behave or valuess of bacteria.) Chromium is found in various oxidation states, very significant of them are trivalent and hexavalent forms. Former is less toxic and more mobile t han the latter. Various bacteria have potential to convert hexavalent chromium t o trivalent form. These bacteria include Pseudomonas and Acintobacter (Farag and Zaki, 2010), Serretia rubidaea (Zhang and Li, 2011), Pseudomonas corrugatta and Pseudomonas mendocina (vitti et al., 2008), P.putida and E.coli (Ackeley et al. , 2003), Streptomyces (Polti et al., 2011), Pediococcus pentosaceus and S. aureu s (Ilyas et al., 2011) and many other bacteria can reduce hexavalent chromium. I TS A KIND OF INTRO I SUGGEST DONT ADD this red highlighted above para. The present research work comprises of the exploitation of potential of pseudomonas sp. isolated from tannery effluent to reduce hexavalent chromium. Fr om three different sites sample was collected including, Rohi Nala, Steel indust ry and Kasur tannery. From these samples three strains were selected namely IIBCr1, IIB-Cr2, and IIB-Cr3. these strains were selected on the basis of their pot ential to resist metal and perform bioreduction. The sites were selected on the basis of having higher chromium contamination in effluent. These strains could tolerate chromium as IIB-Cr1, IIB-Cr2.?? and IIB-Cr3 could tolerate up to 85+_5 g/ml. Wei-hua et al studies the chromium tolerance capacity o f P.auroginosa was 40mg/L (Wei-hua et al., 2009). Resistance to toxic metals is may be due to bioaccumulation, low molecular weight proteins and biotransformati on. (Summers, 1978; Silver and Mishra, 1988). Add one or two more papers showin MIC. The alkaline industrial effluent having several ions in it contains bacteria res istant to several metals like Cd, Zn, As, Cu etc ( Shukla et al., 2007).These st rains have ability to resist arsenic, lead, cadmium, zinc, mercury and copper. T he resistance is shown at variable concentrations. IIB-Cr1showed the resistance to Pb at 800 g/ml, to Cd at 1050 g/ml, to Hg at 200 g/ml, to Cu at 800, and to As a t 500 g/ml. IIB-Cr2 has MIC of Pb at 800 g/ml, Cd at 1050 g/ml, Cu at 700 g/ml, Hg at 200 g/ml, and As at 500 g/ml. IIB-Cr3 has MIC of Pb at 800 g/ml, Cd at 950 g/ml, Cu at 700 g/ml, Hg at 200 g/ml, and As at 500 g/ml. The ability to tolerate cadmiu m, lead and copper was relatively higher than rest of metals. (I think mention o nly the higer value resistance and lower value. And add a few papers showing the resistance to some some other metalsit is commoly given in many papers.) Chromium resistant bacteria isolated from soil contaminated with tannery effluent were gram positive in behavior (Konopka et al., 1999). The behavior of the isolated strains was checked which was gram positive for all 3 strains( sam jh nai ai mjheadd something else.) . Shukla et al studied that the metal toxicity was related to the antibiotic res istance. Majority of bacteria was resistant to Ampicillin and tetracycline while

mild resistance to streptomycin as also shown (Shukla et al., 2007). Our isolat es showed resistance against Ampicillin, tetracycline and erythromycin while sen sitive against trimethoprim. The optimum temperature for chromium resistant bacterial isolates was de termined and it was 36.5C, 37C and 39C for IIB-Cr1, IIB-Cr2 and IIB-Cr3 respectivel y. He et al., (2009) studied that bacillus cereus grows under aerobic conditions and has the potential of chromium reduction (He et al., 2009). All the isolates were capable to grow in the presence of oxygen (its dont matches with optimum te mperature because it is describing aerobic conditions). Ilyas et al., 2011) stud ied the effect of temperature on P.auroginosa that the optimum temperature was b etween 35-40C. The growth increased from 35 C and decreased at 45 C. The bioreducti on of hexavalent chromium was seen between 25-45C (Ilyas et al., 2011). The growth of bacteria is also mediated by change in pH. Ilyas et al., ( 2011) studied that pseudomonas sp. had the optimum pH from 6-9 (Ilyas et al., 20 11) line k start pe b year likhtay hain.. The isolated strains IIB.Cr2 exhibited optimum pH 7.3. (baki strains ke pH ka b btao)Increased in time of incubation a nd pH up to certain limit accelerated the Cr-reduction potential of bacteria (Fa rhana and Malik, 2011). In all the isolated strains the growth rate increased wi th increase in time up to 8hrs. There are several procedures of chromium reduction involving bioaccumula tion, biosorption and enzymatic reduction (Srinath et al., 2002). Our study does nt focus on the mechanism of chromium reduction but the present work will be very helpful in determining the potential of bioreduction of metal resistant bacteri a. (shouldnt it be added with metal estimation paragraph..?? becoz metal estimatio n b tu bioaccumulation ya absorption in bacteria k bad hoti ha)* In pseudomonas and bacillus sp. plasmid was responsible for resistance ( Shukla et al., 2007). Corynebacterium has also plasmid mediated resistance (Vitt i et al., 2003). Add papers which show that Cr resistance gene is on plasmids. The isolation and transformation of plasmid was done and transforma tion was carried out in DH5. Then the cells were grown on IPTG agar media contain ing X-gal. White colonies were selected. Bioremediation was again done using tra nsformed cells. *The chromium accumulation was determined using metal estimation of dige sted cell pallets via concentrated nitric acid. The atomic adsorption spectropho tometer was used for this metal estimation. Add papers showing how much metal wa s processed or accumulated by any other bacteria. The SDS and protein profiling showed the isolates were pseudomonas sp.( NOSDS se nai sequencing se pta chala tha kuch aisay papers add karo in which ur ge nus type bacteria are isolated bot necessarily same species as urz. In SDS you must tell that heavy metal induces some proteins that confer reistanc e to bacteria against contamination etc. The percentage of chromium bioreduction buy IIB-Cr1 was 50%, IIB-Cr2 was 75 % an d IIB-Cr3 was 60%. Arrange paras according to the sequence of ur results or methods. And maam asked me for 4 pages at my time, for discussion.