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ISOLATION AND CHARACTERIZATION OF CHROMIUM REMOVING BACTERIA3.2 Isolation of micoorganisms from theeffluent contaminated soilThe chromium resistant bacteria wereisolated from the effluent treated soil byserial dilution method, each isolated colonieswere picked up and were sub-cultured. Thebiochemical characterizations were carriedout to identify the microorganisms. Cr (VI)resistant bacteria were isolated on the basis ofgrowth in Cr (VI) enriched medium [7]. Allthe soil samples yielded a large number ofcolony-forming units/mL (cfu/mL). Theserial dilution method indicated the differentcolonies in dilution of 10 -5 were found to beGram positive bacteria and gave positiveresult for catalase, starch hydrolysis andgelatine liquefaction tests and negative resultfor IMViC test indicating it to be Bacillus sp.3.3 Optimum growth characteristics ofchromium resistant bacteriaIn the present study, the optimumtemperature for chromium resistant bacteriaBacillus sp, was found to be 37°C (Figure 1).The chromium resistant bacteria, Bacillus sp,showed maximum growth at pH 7 (Figure 2).The growth curve of isolated microorganismin the presence of chromium (K 2 Cr 2 O 7 ) 10µg/mL was compared with the control (i.e nometal ions were added). There was nosignificant difference between control andchromium treated culture (Figure 3). But thegrowth of the isolate was decreased in after18 hours in presence of chromium.3.4 Tannery effluent contaminated soilBoth the control and effluent contaminatedsoil samples were analysed for essentialparameters (Table 2) and it is evident that theeffluent contaminated soil has a greateramount of mineral and metal content than thecontrol soil. Chromium reducing capability ofbacterial isolate was analysed by addingPotassium dichromate (K 2 Cr 2 O 7 ) at 10 µg/mLin the culture medium. The isolated Bacillussp was found to reduce 85.9% of chromiumfrom the medium within 96 h. It was alsocapable to reduce chromium (10 µg/mL) by358%, 68.9% and 74.9% from the mediumafter 24, 48 and 72 hours respectively (Figure3). The heavy metal degradation capacity ofBacillus sp was observed by well diffusionmethod (metal content ranging from 0.5, 1.0,2.0 mg/mL) respectively. The degradationcapacity of Bacillus sp was found to bemaximum at 2.0 mg/mL for chromium,copper, nickel, zinc and cadmium. The zoneof degradation was found to be highest innickel. The heavy metal degradation capacityof Bacillus sp showed maximum resistanceagainst nickel, and the reduction for otherheavy metals was in the order of Ni > Cr >Cu > Zn > Cd.[IV] DISCUSSION4.1 Physiochemical properties of EffluentThe effluent released from tannery industrywas turbid, brown in colour and had anoffensive odour. The colour of the effluentmight be due to the presence ofbiodegradable and non-biodegradable highmolecular weight organic compounds andhigh amount of inorganic chemicals likesodium and chromium used during theprocessing and the odour may be due toputrefaction of the organic residues from theprocessed skin and hides. The yellowishbrown colour might be hindering thepenetration of sunlight causing depletion inthe rate of oxidation process [37;21]. Theturbidity of the effluent might be due to thedischarge of high concentrations ofcarbonates, bicarbonates and chlorides ofcalcium, magnesium and sodium [8].The normal pH range of water should bebetween 6.0 and 8.0 [4]. The effluent had ahigh pH when compared to normal waterindicating the alkaline nature of the effluentSmrithi A and Usha K 647
ISOLATION AND CHARACTERIZATION OF CHROMIUM REMOVING BACTERIAdue to the presence of high concentrations ofsalts of sodium, potassium, chromium etc.[33].The presence of higher level of totalsuspended solids and total dissolved salts inthe effluent might be due to the presence ofinsoluble organic matter from the animal skinand unused inorganic salts used for tanning[19]. The record of high COD might be dueto the presence of oxidizable organic matterfrom the animal skin [29;21].From the study it was clear that highcarbonate and bicarbonate contentcontributes to the total alkalinity of thesample [3]. The level of sodium andpotassium in the tannery effluent was foundto be 2300 mg/L and 600 mg/L respectively.Chromium and sulfide are among the mosthazardous components of the tannerieseffluent. The use of excessive amount ofthese chemicals in tanning process gives riseto their high concentrations in the effluent.Chromium VI is known to cause cancer. Therecommended limit for maximum amount ofchromium in the effluent samples is 1.0 mg/L[6].4.2 Effluent Degrading MicroorganismsThirty four bacterial strains were isolated,identified and characterized, which belongedto different bacterial genera, dominant beingBacillus, Micrococcus and Lactobacillus spp[30;12]. The most suitable temperature for Crresistant bacterial isolate was found to be 37°C. Bacterial Cr (VI) reduction was found tobe maximum at 25 to 30 °C for five bacterialisolates and higher temperature (above 37°C) severely retarded Cr (VI) bioreduction[5]. In a similar study, the optimum pH forthe growth of Cr resistant bacteria wasreported as pH 7 to 7.8. But Cr forms aresoluble over a wide range of pH andgenerally mobile in soil-water systems [34].The optimum initial pH was 7.0 to 9.0 whichcorresponded to a final pH of 6.8 to 6.2.Decrease in culture pH as a result of bacterialgrowth was generally noted. The growthcurve pattern of the isolate was notsignificantly different from that of the controlinitially, but, the growth of the isolatedecreased in presence of Cr 6+ [23].Several Gram positive bacteria were alsoknown to reduce Cr (VI) including severalmembers of the genera Bacillus sp [7].Chromium resistant bacteria capable ofreducing chromate have been reported fromchromium polluted environment and theywere showing resistance in lowerconcentration [18;2]. B. subtilis reducedchromium (VI) under aerobic conditions.This might be due to the presence ofchromium reductase enzyme. Similarchromium (VI) reduction has been reportedby B. coaguratlans. Accumulation ofchromium Cr (VI) by B. circulans has alsobeen demonstrated [26].Pseudomonas strains isolated from metalcontaminated soil harboured resistance tochromium (III)[17]. Reduction of chromiumin industrial effluent was observed inPseudomonas aeruginosa strain isolatedfrom tannery effluent[14]. Bacterial Cr (VI)reduction can occur under both aerobic oranaerobic conditions in presence of differentelectron acceptors such as oxygen, nitrate,sulphate and ferric iron, but the suitablecondition for Cr (VI) bioremediation areaerobic at higher Cr (VI) concentrations andanaerobic at lower Cr (VI) concentrations[28]. It was concluded that bacterial coloniesof Bacillus strain can grow successfully inchromium containing medium as it cantolerate dichromate [24]. It was concludedthat Pseudomonas has the ability to degradeheavy metals present in industrial effluent[31]. It was reported that Aspergillus nigerSmrithi A and Usha K 648
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