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ISOLATION AND CHARACTERIZATION OF CHROMIUM REMOVING BACTERIAzinc, cobalt and chromium. Among heavymetals chromium is the major pollutant of theleather tanning industry and is toxic to plantsand animals around the environment [9].The damage to the environment by thehazardous tannery effluent is becoming anacute problem in several countries. Thechrome tanning process results in toxicmetals, especially chromium III passing towaste water and are not easily eliminated byordinary treatment process [13]. Tannerywaste waters are mainly characterized byhigh salinity, high organic loading andspecific pollutants such as chromium [10].The industrial effluent released directly orindirectly into natural water resources, mostlywithout proper treatment, poses a majorthreat to the environment.Among the different forms of chromium, thehexavalent chromium Cr 6+ is the most toxicand carcinogenic due to its high solubility inwater, rapid permeability through biologicalmembranes and subsequent interaction withintracellular proteins and nucleic acids. Theheavy metals in general cannot bebiologically transformed to more or less toxicproducts and hence persist in the environmentindefinitely [35]. They are significantly toxiceven in small amounts and can cause diseasesin humans and animals as they causeirreversible changes in the body, especially inthe Central Nervous System [26]. Soilcontamination by heavy metals is oftenirreversible and may repress or even kill partsof the microbial community and it isgenerally assumed that the exposure to metalsleads to the establishment of a tolerant/resistant microbial population [32].Microorganisms play a significant and vitalrole in bioremediation of heavy metalcontaminated soil and wastewater.Indigenous soil microbes appear well suitedfor Chromium (VI) transformation in highlycontaminated soil and may accumulatechromium within its cells by adaptation to thehigh concentration of the metal. Very stablefinal chromium forms can be achieved as aresult of microbial activity, with minimal riskof re-release of Cr (VI) [22]. In this paper aneffort has been made to remove heavy metalsfrom tannery effluent using microorganisms.[II] MATERIALS AND METHODS2.1 Tannery EffluentThe effluent was collected from a selectedleather processing industry situated atDindigul in Tamil Nadu, India at weeklyintervals for five weeks, pooled together andstored at 4 °C for analysis. The collectedtannery effluent was analyzed forphysicochemical properties like colour,odour, turbidity, pH, total suspended solids,total dissolved salts, chemical oxygendemand (COD), biological oxygen demand(BOD) [1], carbonate, bicarbonate, sodium,potassium [20], chromium, copper,cadmium, nickel and zinc [1].2.2 Isolation of the microorganism fromtannery effluent contaminated soilFor isolation of chromium resistant bacteria,1.0 g effluent treated soil sample wasdispersed in 100 mL of the sterile distilledwater. A serial dilution was made up to 10 -5from the effluent treated soil sample. 100 µLof each dilution was spread on to nutrientplates containing 10 µg potassiumdichromate (K 2 Cr 2 O 7 ) per mL of themedium. The growth of the bacterial colonieswas observed after 24 h of incubation at 37°C. It was subcultured on nutrient agar platecontaining 10 µg of potassium dichromate(K 2 Cr 2 O 7 ) per mL. Biochemical tests likeGram staining [27], dextrose fermentation,sucrose fermentation, IMViC, catalaseactivity, starch hydrolysis [11] and gelatinSmrithi A and Usha K 645
ISOLATION AND CHARACTERIZATION OF CHROMIUM REMOVING BACTERIAliquefaction [15] were carried out to identifythe isolated microorganisms.2.3 Optimum growth conditionsFor the determination of optimumtemperature for the growth of the isolatedbacteria, test tubes containing 5 mL nutrientbroth were taken in four sets, autoclaved andinoculated with 20 µL of freshly preparedculture of the isolate. The four sets of tubeswere incubated each at 25, 30, 37, 45 °Crespectively. After an incubation period of 12hours, the absorbance was measured at 600nm. For the determination of optimum pH,test tubes having 5 mL nutrient broth weretaken in 9 test tubes and then, the pH wasadjusted to 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0,8.5, and 9.0. The test tubes were inoculatedwith 20 µL of freshly prepared culture of theisolate. After 12 hours of incubation, theabsorbance was measured at 600 nm.2.4 Growth rate of bacterial isolates withand without chromiumGrowth curves of bacterial isolates weredetermined with 10 µg K 2 Cr 2 O 7 per mL andwithout K 2 Cr 2 O 7 (control). For bacterialisolates, 50 mL nutrient broth was taken inone set consisting of three flasks, autoclavedand then inoculated with 20 µL of the freshlyprepared inoculum. These cultures wereincubated at 37 °C in a shaker at 30 rpm. Theabsorbance of the cultures was measured at600 nm at intervals of 2 h up to 24 h afterinoculation.2.5 Removal of chromium by bacterialisolatesIn order to determine the ability of bacterialisolate to reduce Cr 6+ to Cr 3+ ,spectrophotometric method was used. 5 mLsamples were taken from the culture after 24h, spun down at 10,000 rpm for 5 min andthe supernatant was used for the estimationof chromium left in the medium. From this1.0 mL sample was taken and diluted with 9mL of distilled water in the test tubes. Themixture was kept at room temperature for 10min and OD was taken at 600 nm.2.6 Removal of heavy metals by isolatedbacterium20 mL of the sterile medium containingnutrient agar 15 g, K 2 HPO 4 0.5 g, K 2 PO 4 1.5g, glucose 0.05 g and distilled water to 1000mL was poured into sterile petriplates andwas allowed to solidify. Then each isolatedmicroorganism was swabbed on the medium.Four wells were made on each petriplateusing well puncture. Different concentrations(0.5, 1.0, 1.5 and 2 mg/mL) of metals (Cu,Cd, Cr, Ni, and Zn) were made and loaded onthe wells. Then the plates were incubated for24 h at 37 °C[3] RESULTS3.1 Tannery EffluentThe collected leather tanning industrialeffluent was assessed for its physicochemicalproperties and toxic metal levels (Table 1).The effluent sample analyzed had a pH valueof 10.5. The total suspended solids (2300mg/L) and total dissolved salts (12,900 mg/L)in the effluent sample were found to be veryhigh when compared to BIS standards. CODand BOD in the selected effluent samplewere found to be 3180 mg/L and 1300 mg/Lrespectively.In the effluent sample analyzed, the presenceof carbonates and bicarbonates were found tobe very high, 7250 mg/L and 10,238 mg/Lrespectively. Chromium is the widely usedheavy metal in tannery industries and wasfound to be 179 mg/L in the effluent. Othermetals like cadmium, chromium, copper,nickel and zinc were present at levels of 4.81,179, 64.32l, 132, and 171 mg/L respectively(Table 2).Smrithi A and Usha K 646
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