Removal of Cr(VI) from Aqueous Solutions by Growing and Non-growing Populations of Environmental Bacterial Consortia

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1 Glol Journl of Environmentl Reserch, (): -7, 007 ISSN IDOSI Pulictions, 007 Removl of Cr(VI) from Aqueous Solutions y Growing nd Non-growing Popultions of Environmentl Bcteril Consorti J. Kder, P. Snnsi, O. Othmn, B.S. Ismil nd S. Slmijh Fculty of Science nd Technology, Universiti Kengsn Mlysi, UKM Bngi, Selngor D.E. Mlysi School of Science nd Mthemtics, INTI College Mlysi, 7800 Bndr Bru Nili, N. Semiln, Mlysi Astrct: An environmentl cteril mixed culture, Consorti Culture (CC), ws employed in this study to ssess nd demonstrte the extent of Cr(VI) reduction nd uptke y cells t concentrtions rnging from to 00 mg L. Growing (ctive) cells of CC displyed higher overll men Cr uptke (50.74 mg g DW), with elevted men Cr reduction ( mg g h ) nd ccumultion rte of mg g h compred to non-growing (inctive) cells over period of 96 h. Removl of Cr from the culture medium y CC ppers to e ttriuted to ccumultion or cellulr uptke rther thn reduction to Cr(III). Key words: Chromium Bcteril consortium Hevy metl INTRODUCTION chemicl reduction followed y precipittion, dsorption on col, ctivted cron, lum, kolinite In recent yers, the use of iologicl methods nd fly sh nd on ion exchnge nd chelting resins. prticulrly microes to remove metllic ions from These processes re energy nd cost intensive nd liquid effluents hs received greter ttention over the inefficient t low concentrtions, producing y-products conventionl physico-chemicl methods. Hevy metl of toxic sludge [, 5]. iosorption studies undertken thus fr concentrte on Studies on chromte (CrO 4 ) removl in Cr-resistnt the ility of pure microil species to remove ctions cteril systems hve indicted the onset of reduction nd there is very little documenttion on the use of nd the possile uptke of the nion y the orgnisms mixed cteril consortium encompssing Grm positive [, 6, 7]. Three possile wys for CrO 4 removl from nd Grm negtive cultures [, ]. Consorti cultures re Cr contminted wste wters hve een proposed; etter equipped in terms of metolic nd removl i) reduction to Cr(III) y cteril cells nd precipittion cpility. Moreover, the consorti would e more outside the cell nd into the solution, ii) ccumultion of suitle for field ppliction tking into ccount the Cr (oth hexvlent nd/or trivlent forms) y the cell spect of competition nd survivl. iomss or iii) comintion of these two processes [, 3]. Cr is present in wste wter from ctivities such There is no conclusive reltionship etween reducing s metl finishing, metl plting, petroleum refining, ility, resistnce nd metl ccumultion cpcity nd chrome tnning, textile mnufcturing nd wood they pper to e governed y the microes involved processing to nme few [, 3, 4]. Hexvlent chromium, [, 8]. A comintion of metl resistnce nd enhnced which occurs s chromte (CrO 4 ) nd dichromte metl removl cpcity would e n dvntge for the (CrO 7 ) in nturl wters re very solule nd re development of good iosorent. reported to e crcinogenic nd mutgenic to living This work reports n investigtion on the extent of orgnisms [4]. When reduced to the trivlent form chromte reduction nd chromte uptke/ccumultion (Cr(III)) they re reltively insolule, more stle nd in oth growing nd non-growing mixed cteril less toxic thn the prent compounds. Conventionl popultions which hve een isolted from the tretment methods for toxic chromte removl include environment. Corresponding Author: Dr. B.S. Ismil, Fculty of Science nd Technology, Universiti Kengsn Mlysi, UKM Bngi, Selngor D.E. Mlysi

2 Glol J. Environ. Res., (): -7, 007 MATERIALS AND METHODS Non-growing (inctive) cells: Tests were done in sterile ddho suspended with pproximtely 0. g L Bcterium consorti: The cteril consortium, termed dry weight iomss. s Consorti Culture (CC), consists of six Grm negtive (Pseudomons sp., Serrti sp., Flvocterium sp., Metls: Cr(VI) ws dded to the medium to set the Chryseomons sp., Xnthomons sp. nd Agrocterium desired concentrtion from the concentrted sterile stock sp.) nd three Grm positive (Bcillus sp., Arthrocter solution of KCrO 4 which ws prepred with NANOpure sp. nd Micrococcus sp.) environmentl isoltes. Bcteril deionised wter. No precipittion occurred nd the isoltes include comintion of strins isolted from solution chemistry of Cr shows Cr (VI) is dominnt s the point nd non-point sources in res relted to metl- chromte or dichromte species t ph 6.8±0.. sed ctivities. The isoltes were screened intensively nd developed from pool of mixed cultures [9]. Effect of Cr(VI) on microil growth: To ssess the effect of Cr (VI) on cell growth, CC were inoculted into 0 ml of Medi nd growth conditions: The Consorti Culture (CC) NB contining Cr (VI) t, 0, 0, 40, 60, 80, 00 mg L. ws mintined t room temperture in sl medi Suspensions of growing cells were incuted s tch contining (in g L ): yest extrct (0.5), peptone (0.5) cultures for 96 h. Growth of the cteri ws monitored nd NCl (8.5) with fortnightly medi refreshments. periodiclly (0,, 4, 6, 8, 0,, 4, 36, 48, 60, 7, 84 nd Two types of culture i.e. growth culture nd enrichment 96 h) y mesuring the OD t 600 nm [7]. culture were initited nd employed for inocul preprtion. The two cultures would provide the est Metl removl nd kinetic studies of Cr(VI): Btch working inocul, s growth culture would ensure cultures contining vrying concentrtions of Cr (VI) mximum iomss production while enrichment culture (, 0, 0, 40, 60, 80 nd 00 mg L ), t n initil ph of 6.8 (initilly enriched with mg L of ech P (II), Cu (II) were incuted t room temperture. The mount of nd Cr (VI) nd susequently incresed to 0 mg L ), totl Cr (in cells nd medi) nd Cr(VI) (in medi) were would generte metl cclimted cteril community. determined fter 0, 6,, 4, 36, 48, 60, 7, 84 nd 96 h. The All tests were crried out in sttionry mode t room mount of Cr(VI) reduced to Cr(III) is given y: totl Crtemperture (8-30 C) with n initil ph of 6.8±0.. Cr(VI) in the solution [0]. The mount of Cr(VI) reduced Microil growth ws monitored y opticl density (OD) to Cr(III) ws compred to the mount of Cr ccumulted t 600 nm. in the cells (Cr mss lnce) within the time tested nd the rtes y which these processes occur in oth cell Inocul preprtion: Aliquots from oth growth nd popultions were determined. Metl loding (q p) of enrichment cultures (0.5%,v/v) were inoculted into 0 ml iomss ws clculted using the generl definition q p = nutrient roth (NB) nd incuted t room temperture C p(v)/w, where q pis the metl loding (mg metl g DW) for 48 h. Cell iomss ws seprted y centrifugtion in solution, V (ml) nd C p is the mount of Cr (mg L ) (4000 rpm, 0 min). Pellets were wshed, re-centrifuged contined in the iomss of known dry weight, w (mg) []. nd rinsed twice efore eing re-suspended in either NB Cells were removed y centrifugtion nd were or sterile deionised distilled wter ddho. Cell density digested with concentrted HNO 3 t C for 5-6 h ws stndrdised t OD 600 of (contining pprox. prior to totl Cr determintion. Concentrtions of Cr(VI) 7 0 cells ml ) nd served s the source of inocul. A in the medi ws determined spectrophotometriclly using stndrd curve ws prepred to estimte the iomss the diphenylcrzide method [] nd Cr concentrtions quntity used throughout the study (mg dry weight in the medi nd the cells were mesured y the tomic (DW) plotted ginst OD 600, R = 0.999). For iomss dry sorption spectrophotometer (PE 00B, Perkin Elmer). weight (DW) determintion, pellets were dried in n oven The mount of Cr reduced nd sored onto cells ws for 4-48 h efore eing heted further to 0-0 C for corrected using the pproprite controls to ensure ll Cr 0-5 min until they ttined constnt weight. in the solution ws ccounted for. The totl Cr ccumulted y the cells, the residul Cr(VI) nd Cr Growing (ctive) cells: Tests were done in NB with n concentrtions in the solution were clculted inocul size of % (v/v). After 4 h, cells would rech ccordingly. Suitle controls were included in ech of pprox. 0. g L dry weight iomss. the experiments. 3

3 Glol J. Environ. Res., (): -7, 007 Tle : Men OD t specified intervls (up to h) for CC growing cells in NB supplemented with Cr (VI) 600 OD fter time (h) Cr (VI) (mg L ) Control Tle : Men OD t specified intervls (over 96 h) for CC growing cells in NB supplemented with Cr (VI) 600 OD fter time, h Cr (VI) (mg L ) Control : Denotes significnt effect y decrese in growth in comprison to control t = 0.0 : Denotes significnt effect y decrese in growth in comprison to control t = 0.05 RESULTS AND DISCUSSION or/nd intrcellulr ccumultion. Nies [] suggested tht chromte is ctively trnsported into the cell y Effect of Cr (VI) on microil growth: The effect of Cr the sulfte trnsport system nd other dditionl (VI) on cell growth over period of 96 h is presented in systems which possily re relted to induction or Tle nd. The onset of cell growth (up to h) t repression of certin ion trnsport systems. CC ws low Cr (VI) concentrtions (-0 mg L ) ws comprle shown to e resistnt s it could very well tolerte Cr(VI) to tht of the control ut declined with incresing Cr concentrtions of up to 00 mg L. Mechnisms for concentrtions. Cr t 0-00 mg L significntly inhiited chromte tolernce in cteri include reduction, cteril growth (p<0.0). This is indicted y longer lg methyltion, precipittion t cell surfce, lockge or periods of etween to 6 h t higher Cr(VI) modified cellulr uptke y ltered pthwy nd concentrtions compred to pproximtely 8 h t lower susequent removl from the cytoplsm y efflux pump concentrtions. Between -0 mg L Cr(VI), mximl [6, 3]. growth (OD ) ws chieved within 4 h; t higher Cr(VI) concentrtions, delyed growth ws oserved. Metl removl nd kinetic studies of Cr(VI): The The inhiitory effect of Cr(VI) ws not significnt reduction of Cr (VI) to Cr (III) nd the ccumultion of Cr (p>0.05) in the ltter stge of growth ( 4 h) for ll in the -00 mg L for oth cell popultions re given in concentrtions tested except 60 mg L. The long lg Fig.. In growing cells Cr ccumultion ( mg L ) period oserved in the erly phses of growth nd ws higher thn its reduction ( mg L ) (p<0.05). prticulrly t elevted Cr(VI) concentrtions, ws Similrly, non-growing cells lso exhiited higher proly due to the dpttion of the popultion to the ccumultion ( mg L ) compred to reduction environment. In order to grow in chromte contining ( mg L ) (p<0.05). Accumultion of Cr y medi, cteril system would need to develop growing cells ws slow in the erly stges i.e. 8-0 h detoxifying tool y either reduction or exocellulr which correlted with the slower growth trend s hs een 4

4 Glol J. Environ. Res., (): -7, 007 Fig. : Cr reduction nd ccumultion in growing (A) nd non-growing (B) cells t ( - mg L ; -0 mg L ; -0 mg L ; X-40 mg L ; -60 mg L ; -80 mg L ; +-00 mg L ) initil concentrtion over 96 h Tle : Rte of ccumultion nd reduction of hexvlent chromium, Cr (VI) in the initil concentrtion rnge of -00 mg L nd cell concentrtion s function of men contct time (96 h) Consorti culture Men Cr reduction Men Cr Men Cr Men Cr (CC) cells rte (mg g h ) ccumultion rte (mg g h ) reduced (mg L ) ccumulted (mg L ) Growing Non-growing Tle : Overll system chrcteristics for the removl of Cr (VI) in the initil concentrtion of -00 mg L over 96 h Consorti culture Men cell density Men Cr uptke, Men ph Men of (CC) cells (mg DW) q p (mg g DW) chnges unccounted Cr (mg L ) Growing Non-growing descried erlier. Within the first 4 h, the mount of Cr compred to Cr reduction ( mg g h ) in (VI) sored y growing cells ws comprle to tht of oth popultions over the 96-h period. Generlly, the non-growing cells. However, ccumultion of Cr in rtes nd mount of Cr reduced were dependent on growing cells still incresed fter 4 h compred to non- cell ctivity s growing cells reduced nd ccumulted growing cells s ccumultion reched plteu within fr more Cr thn the non-growing cells. The incresed 4 h indicting iomss sturtion. Similr trends were mount ccumulted ws more pprent t higher noted t ll Cr(VI) concentrtions tested throughout the concentrtions (00 mg L ), i.e. 6.4 mg L compred to 96 h period..8 mg L reduced y growing cells fter 96 h. In terms Tle shows tht the rte of Cr ccumultion y of the removl process, it ws ovious tht ctive cells ctive cells ws lso fster ( mg g h ) were required for Cr reduction s the men Cr reduced 5

5 Glol J. Environ. Res., (): -7, 007 Tle 3: Suggested model for the removl of Cr (VI) Consorti culture (CC) cells Model * Correltion coefficient Growing cells C cc = t pH Citot-0.985C ftot w R =.000 C red = t-0.5pH + 0.9Citot-0.95Cf(VI) -0.57w R = Non-growing cells C cc = t pH Citot-0.996Cftot w R = C red = t pH Citot C f(vi) w R = 0.84 C cc: Cr ccumultion ility, (mg L ), C red: Cr(VI) reducing ility, (mg L ), C itot: initil totl Cr concentrtion, (mg L ), C ftot: finl totl Cr concentrtion, (mg L ) t t, C f(vi) : finl Cr(VI) concentrtion, (mg L ) t t, ph: solution ph, t: time, w: iomss quntity, (mg dry weight), ll prmeters re significnt t = 0.05 ws only 0.30 mg L in non-growing cells s opposed fter reduction is lso possily sored y the iomss to.80 mg L in growing cells. Lower uptke t higher rther thn excreted outside the cell. The use of n concentrtions nd t extended periods my e due to cclimted mixed culture such s CC would redily sturtion of the iomss, given the miniml iomss provide n undnce of ctive sorption sites to fcilitte quntity employed in this work. On the other hnd, inding or complextion. The consorti would lso e possile inhiition of iomss ctivity y prolonged well-equipped nd evolved in terms of cellulr chromte toxicity would lso e reson s it decreses defense/resistnce mechnisms due to continuous Cr ctive cell popultion in the medium s well s nutrient exposure nd possile synergistic reltion mong the depletion [4]. This reveled tht Cr uptke is more ville strins. dominnt for Cr removl compred to the insignificnt Further investigtions such s lortory scle Cr reduction in oth cell popultions. CC exhiited metl removl fesiility studies need to e crried out high men of Cr loding of mg g for growing on the Consorti Culture (CC) efore they cn e cells nd 3.66 mg g for non-growing cells (Tle ) exploited s iosorents for the detoxifiction nd nd this is comprle nd etter thn the other systems removl of Cr from wstewter. Aprt from the ove, descried [5-7]. Aiotic nd experimentl Cr losses moleculr studies will most proly elucidte in more were insignificnt nd smll ph fluctutions oserved detil the mechnisms involved. might e due to cell ctivity nd possile nion exchnge, - ssumed to e with OH, s the vlue incresed. ACKNOWLEDGEMENTS Using multiple regression nlysis nd the ANOVA test (Tle 3), Cr(VI) removl model ws developed This study is funded under IRPA to descrie optiml ccumultion (C ) nd reduction EA6 from the Ministry of Science, Technology nd cc (C ). This model took into ccount independent vriles Innovtion, Mlysi. red i.e. initil totl Cr concentrtion (C ), finl totl Cr itot concentrtion (C ftot), finl Cr(VI) concentrtion (C f(vi) ), REFERENCES iomss quntity (w) nd solution s ph (p<0.05). Both the proposed models gve good correltion with the. Bdr, U., N. Ahmed, A.J. Beswick, P. cell popultion tested (R >0.8). This model cn very Pttnpipitpisl nd L.E. Mcskie, 000. well e used s guide for further studies involving Reduction of chromte y micro-orgnisms isolted scling-up. from metl contminted sites of Krchi, Pkistn, CONCLUSIONS. Biotechnol. Lett., : Krtochvil, D. nd B. Volesky, 998. Advnces in the iosorption of hevy metls. Titech., 6: Bsed on ll the tests crried out, it cn e seen tht 3. DeLeo, P.C. nd H.L. Ehrlich, 994. Reduction of CC cn thrive in solutions contining up to 00 mg L Cr hexvlent chromium y Pseudomons fluorescens nd demonstrte good metl removl cpility. Growing LB300 in tch nd continuous cultures. Appl. (ctive) cells hd higher uptke/ccumultion cpcity Microiol. Biotechnol., 40: (30-50%) nd reducing ility (40-80%) compred to non- 4. McComish, M.F. nd J.H. Ong, 988. Trce metls. growing (inctive) cells. Generlly, uptke/ccumultion In: Bodek, I., W.J. Lymn, W.F. Reehl nd D.H. ws well defined (50-90%) in oth cell popultions thn Rosenltt, (Eds.). Environmentl Inorgnic reduction. This suggests tht Cr cn e sored y the Chemistry: Properties, Processes nd Estimtion iomss in the hexvlent form nd the Cr(III) formed Methods. New York: Pergmon Press Inc. 6

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