Indian Journal of Ex perimental Bio logy Vol. 4 1, September 2003, pp. 972-985

Bioremediation of chromium contaminated environments

Sara Parwin Banu Kamaludeen, K R Arunkumar, S A vudainayagam & K Ramasamy*

Fermentation Laboratory. Tamil Nadu Agricultural Uni vers ity, Coimbatore 64 1 003, India

Bioremed ia tion is the most promising and cos t effecti ve techno logy widely used nowadays to c lean up both so il s and wastewaters containing o rganic o r inorgan ic contaminants. Discharge of chromium conta ining was tes has led to destruc ti on of many ag ri cu ltural lands and water bod ies . Utili sa ti on o f chrom ium(Cr) reduc ing microbes and their products has enhanced the effic iency o f the process of de tox ifica ti on of C r(V I) to C rUll ). T hi s rev iew focuses mainly on the cu rrent technologies prevalent fo r remedia ti on li ke natura l atte nuati on, anaerobic packed bed bio reactors (us ing li ve cell s, C r(VI) red uctases or the ir byproducts) and usc of engineered microorgani sms. T rea tment of wastewaters by biosorpt ion o r using biori lms and immobili zed microbia l cells a re a lso d isc ussed.

Key words: Bioremediati on, C hromium contaminati on, C hromium reducing mi crobes, Eng ineered microorgani sms

Disposal of industrial and urban wastes to soil and water bodi es has led to di sastrous consequences to these ecosystems. Due to the excess loading of these wastes beyond their self cleaning capac ities, these ecosystems has resulted in decreased availability of clean water to drink and normal soil s for crop producti on. Enormous amount of organic and inorgani c wastes from va rious industries has been di sposed di scriminately before the enactment of stringent regul ations in many countries including India. Compared to the organic wastes, inorganic wastes, like heavy metals, pose a great threat, as they cannot be completely removed/degraded from the ecosystem like organi c pes ti cides.

Chromium is an important heavy metal widely used in the metallurgic, refractory, chemica l and tan nery industri es. Chrome pl ating, the deposition of metalli c Cr, imparts a refrac tory nature to materi als rendering them resistant to microb ial attack and flex ible over extended peri ods of time l

. More than 170,000 tons of Cr wastes are di scharged to the envi ronment annuall y as a consequence of industri al and manu fac turing ac ti viti es2

. Of the total Cr used in the process ing of leather, 40% is retained in the sludge, di sposal of which onto land and into water bod ies has led to increased Cr levels reaching as high as 30,000 mg kg-J3

Deta iled assessment of the tannery waste contam inated sites in Tamil Nadu, India and Mount Barker near Adelaide, Australi a revealed the

"Correspond ing aut hor: E-ma il: ramasamytnau @yahoo.com, Fax: 0091-422-243 1672

ex tensive contamination of soil and surface or ground water.4-7

. Although no definite pattern was observed in di ffe rent depths, it was reported that the concentration ranged between 100 mg to 70,000 mg kg-I in surface and subsurface soil s of Tamil Nadu where old tanneries were located. The presence of high concentrations of soil chromium pose significant risks to animals and humans li ving in the vici nity of these sites through dust parti cles during summer rn Adelaide and winter months in India.

Distribution Chromium is a potential so il , surface water, ground

water, sediment and air contaminant. Soi l chromium levels are usually related to the chromium level in the parent material, and except in soils deri ved from serpentine soil materi als the natural background levels of soil chromium are actuall y lows.9. Chromium concentration in different environmental components is shown in Table 1.

Hi gh soil chromium levels are usua lly associated with anthropogenic contamination, mainl y from industri al operations 10. Tanneries is one among the major industries and based on the processes used the compos ition of the sludge vari es as well as the concentration of Cr (Table 2)11.

Speciation of chromium Chromium tox icity and mobility depends on its

ox idati on state. Though chromium can ex ist in ox idation states rang ing from 2- to 6+ 12, onl y chromium (V I) and chromium (111) are normally fo und within the range of p H and redox poten ti als

KAMALUDEEN el at.: BIOREMEDIATION OF CHROMIUM CONTAMINATION 973

Table I -Chromium concentration in different environmental components

Environmental component

Continental crust Soil Fresh water Sea water Drinking water Air samples

Concentration

80-200 mg/kg 10- 150 mg/kg 0. 1-6.0 mg/L 0.2-50.0 mg/L 0.05 mg/L 0.0 15-0.03 mg/m)

Table 2 - Chemical composi tion of tannery chrome sludge from d ifferent countries

Composition of tannery • Sou th USA South s ludge Austra li a India

pH 7.7 1 * 7.8 EC (dS m-I) 3.5 * 3.5 Total organic carbon (%) II 15.4 9.6 Cr (%) 3.04 3.86 0.08 CaCO) (%) 11.7 * * Ca (%) 4 .7 1.8 0.8 Na (%) 4.7 0 .7 * Fe (mg kg-I) 1.1 * * Mn (mg kg· l

) 1.3 * * Cu (mg kg-I) 66.5 * * N (mg kg· l

) * 3 1000 4300 P (mg kg-I) 4032 * 5 100 K (mg kg· l

) 3549 1900 40

*Not reported

concern of various environmental systems (Fig. 1). The trivalent forms are relati vely immobile, more stable and much less tox ic than hexavalent formsI3.14 . Chromium, based on the phys icochemical conditi ons, occur either as tri va lent , Cr(l II ) or hexava lent , Cr(VI) specIes.

Among thi s the hexavalent species, Cr(VJ), because of its mutagenic and carc inogenic nature, is considered as one of the priority pollutants l5. In the absence of reducing agents, Cr(VI) is soluble and hence mobi le and it pose a great threat to surface and groundwater quality.

Transformation of chromium Chromium is known to undergo various chemical

and biological reac tions in the natural systems that govern speciation of the metal and in turn its environmental behaviour. Important reactions include ox idat ionlred uction, precipitation/dissolution and absorption/ desorption . Both ox idation of Cr(llJ ) and reduction of chromium(V I) can occu r in geologic and aquatic environments.

ChemicaL transformatioll Hexavalent chrom ium is a strong ox idati on age nt

and is readily reduced in the presence of appropriate

electron donors. Rai and Zachara16 reported that Fe(II) actively reduced Cr(VI) and the reaction rate was dependent on the solubility of Fe compound. The presence of organic matter enhanced · Cr(VI) reduction I7.18. Low oxygen status also resulted in reduction of Cr(VI) and Cr(III)1 9.

Microbial transformation The microbial inter conversions of heavy metals

are of prime interest, since most of the heavy metals have entered the environment as a result of new industrial processes. The discovery of microorgani sms that preferentially reduced hexavalent chromium has led to applications in the bioremediation of chromium contaminated ecosystems (Table 3). Bioreduction of chromate can occur under both aerobic2o-22 and anaerobic conditions23,24.

Direct and indirect bioreduction and altered uptake appears to be a function of resistance to chromate. Bioreducti on of chromate OCCUlTed directly as a result of microbial metaboli sm or indirectly by the action of bacterial metabolites such as H2S

20. Some of the importan t microbes involved in chromium reduction are Euglena gracilis, Pseudomonas aeruginosa, Enterobacter cloacae, Pseudomonas fluorescens, etc.

Anaerob ic bacteri al strains wi th accelerated Cr (V I) reducing capabilities have been isolated from chromate contaminated water and sludge23-26. As heavy metals occur naturall y at high concentration in various environmental systems, res istance and tolerance mechani sms are being evolved within microbial communities. Certain Cr(VI) resistant strains like Enlerobacter sp and Pseudomonas sp. reduced Cr (V I) to Cr (Ill ) under anaerobic condi ti ons22.25.27.28 .

Komori el al23 reported that Enlerobacter cloacae strain HOI reduced Cr(VI) anaerobically while grow ing on acetate, ethanol, malate, succinate and glycerol but its reducti on efficiency was decreased significantl y while the organi sm was grown on glucose, molybdate, vanadate and manganese ox ide. It was further recorded that rate of reduction was proportionate to cell density , optimum pH and temperature were 7.0 -- 7.8 and 30-3rc.

Turick et al ,z9 isolated hexava lent chromiu m reducing anaerobes from hexavalent chromiu m contaminated environments. These organisms were capable of red uci ng tox ic and mobile Cr(V I) to less toxic and immobile Cr(l ll ). In add iti on the apparent ubiquity of Cr(V I) red ucing bacteria in soils and sed iments indicated the potential for III SilU

974 INDIAN J EXP BIOl, SEPTEMBER 2003

bioremediation of Cr(VI) contaminated soils and ground water. Defilippi and Lupton30 des igned an anaerobic bioreactor utilizing marine derived, sulphate reducing bacteri a, immobili zed as a biofilm on gravel. Their experiments have suggested that with

Cr citra te

..... ............... .....

Cr (III) preci pitales and pol ymers

citrate

additional clarification, Cr level can be brought down to as low as 0.01 mg L· t

• This is due to the reducti on of Cr(VI) by H2S produced by the sulphate respiring bacteria. The H2S produced by sulphate respiring bacteria in anaerobic systems diffused out into the

'" HCrO; '"

'"

leac hing plants uptake

'" )If adsorption/ '" '" precipita ti on

....... :' SUN ':

.....

Fig. I - Chromium speci ation in natural ecosys tem"

Table 3 - Microbes used in bioremediat ion o f Cr

Mechani sm Organis m

Bi oreduction Pseudol7lonas ambigua G-l

Bioreducti on Sulfate-reducing bacte ri a

Biorcduction Un identified bac teri a l cultures

Bio!'cducti on Chalamydomonas sp

Biosorplion Oscillmoria sp

Biosorplion ArlhrobaCler sp Agrobaclerium sp

Biosorption/Bioreduction Pseudomonas aeruginosa 5 128

Biosorption/ Bioreduction Sulfa te-reduci ng bac teria

Source : modified from l osi et al9

Descripti on and Effect iveness

Cr(VI) concentration was lowered from ISO to 3S mg l ·1 ovcr 36 hr in liquid media

Cr(VI) in water was lowered from II to <0.2 1 mg l ·1 with addit ion of sulphate and ace tate in an anaerobi c bioreactor that produced H2 S, subsequently reduc ing the Cr(V I)

Reduced 40-60% of Cr (VI) added to liquid media at 10 and 25 mg l ·1 approximately 50% at 60 mg l ·1 and 10% at 120 mg l ·1 over 18 hr with no additi onal nutrient suppleme nt

An average of 30% Cr removed from water with initi a l concentrations of 0.2 mg L·1 in an aerobic bioreduction system

Algal c ultures removed 20% of Cr from water spiked at levels of 1-20 mg L·1

Accumulated Cr with increasing concentration gradie nt of Cr (VI) up 400 mg l ·1 (Arthrobacter) and 100 mg l ·1 (Agrobacterium )

Re moved IS-SO% of C r from liquid media spiked with 1,000 mg L·1 Cr (V I) over 82 hr. Addition of g lucose enhanced the removal rate.

100% Cr removal was achieved in water with Cr (VI ) up to ISO mg L·1

KAMALUDEEN el al.: BIOREMEDIATION OF CHROM IUM CONTAMINATION 975

medium and reduced Cr(VJ). The tri valent chromium thus formed react with OH· to form insoluble / immobile Cr(OH)3. This technique is also being evaluated as a possible in SilLi treatment for immobili zation of Cr in chromate contaminated soil s and ground water31.

Bioremediation Bioremediation has been used as a strategy using

microorganisms (i ntroduced or indigenous) for complcte transformation of organic pesticides to harmless end products such as COl and H20. Likewise, microorganisms can transform inorganic pollutants, not necessarily completely, but to compounds with decreased solubility, mobility and toxicity. For instance, as stated in Table 3, microorganisms can transform toxic and reactive Cr(VI) to less toxic Cr(l ll ).

Cr(VI) bioremediation technology A wide range of microorgani sms ex hibits an

exceptional capacity to detoxify Cr(VJ) by convert ing it to less soluble and much less tox ic Cr(lll) . This capac ity is harnessed in bioremediation technology for Cr(VJ) wherein the microbial strai ns are multiplied to des ired populati on and pumped into soil/sediments in reactors to promote Cr reduction. The bioremcdi ation efficiency can be enhanced by supplements wi th organic matter and other nutrients in the water/soil to promote the growth of the introduced mi croorgani sms. The addition of organi c sources to the so il can promote the proliferation of indigenous Cr(Vl )-reducing microorgani sms as well since Cr(VI ) reducers, both aerob ic and anaerobic, are ubiquitous in the so il environment. Losi e l al.9

decontam in ated largc volumes of Cr(VI)­contaminated water by passing it through an organic amended (cattle manure) soil. Indigenous soil microorgani sms augmented by the organi c amendments were largely in vo lved in the reducti on of Cr(VI) in the water, followed by precip itation and immobili sati on of the Cr(lll ) formed . In in SilLi

techniques. nutrients are pumped along with aeration to promote the Cr reduction by aerobic Cr(V I)­rcducing bac teria. Some Cr-red ucing bacteri a and algae ha ve been efficientl y used in the treatment of Cr-rich was te water9.32.3J B ioreactors are cost­effecti ve and are effective for decolltami nation of Cr(VI)-contaminated was te water. However, success has been limited for large sca le decontamination of Cr(V l)- pollulCd complex so il s.

Recently, fo r treatment of so il s enri ched with chromi te ore process ing residue, a technique

involving the use of organic-rich acidic manure along wi th chrome reducing microbes to effecti vely reduce the Cr(VI) in the waste has been developed. This layer is positi oned below the Cr-rich waste and Cr(Vl) leaching out of the waste, is effecti ve ly reduced in the organic layer, thereby preventing f h .. f d 3435 urt er contam1l1atlon 0 groun water· . .

As described by Losi e l al .9, the bioremcdiation of the Cr(V I)-contaminated soil is achieved by either direct or indirect biological reduction . Most of the direct microbial reducti on would be ex pected on surface soils. In the subsurface layers, indirect biological reduction of Cr(VI) involving H2S can be predominant and very effective, especiall y in situations where in situ stimulation of sulfate reducing bacteria is achieved through the addition of sulfate and nutrients. The H2S, diffused into inaccessible soil pores, promotes the reduction of not only Cr(VI), but also Mn oxides, involved in reoxidation of Cr(Ilf). This method has shown some promise for remediation of Cr(V I) contaminated soil s when app lied to an anaerob ic bioreactor system9.

Anaerobic packed-bed bioreactor Anaerobic Cr(Vl)-reducing microorgani sms are

known to be ubiquitous in soil s27. Anaerobic chromate reducing strains have been successfully used for the

d · d d· . f 29.36.37 re uctlon an se Imentatlon 0 tannery wastes . Turick and hi s group have developed an anaerobic bioprocess for Cr(Vl ) red uction using a mixed culture of so il isolates or indigenous microorgani sms in a packed-bed bioreactor containing ceramic packing or DuPont Bio-Sep beads3s

.39

. There is ev idence to suggest that organic contaminants such as aromatic compounds are suitable electron donors for Cr(Vl)

d . 40 Cl . d· . b h re uct lon . lromlUIl1-re uClng micro es may t en be able to simultaneously remedi ate organic contaminants as well.

Scope for engineered microo"ganisms Cr(VI) reduction by a wide range 01

microorgani sms is of environmental and bi otechno­logica l signi ficance. Bioremed iati on of chromate­polluted env ironments often poses two major problems: (i) inability of introduced Cr(V! )-red ucing mi croorgani sms to es tab li sh and functi on at sites polluted with mixtures of contaminants; and (ii ) biodegradation rates not adequate enough to ach ieve acceptab le residue levels within an acceptable time fralne. Seve ral strategies have been proposed to enh ance the ra tes of bioremediation of pollutants in

976 INDIAN J EXP BlOL, SEPTEMB ER 2003

such inhospitable environments. One of the approaches is to develop eng ineered novel strains with increased Cr(VI)-reducing effi c iency for such situati ons. Recently , Gonzalez4 1 cloned two bacteri al genes encoding different soluble chromate reductases (Class I and Cl ass II) that reduce Cr(VI) to Cr(lll) . Each class has several c lose struc tura l homologs in o ther bacteri a . Fi ve of these prote ins, overproduced in pure form, ~ould reduce chro mate and quino nes. C lass Il prote ins could a lso reduce nitroaromatic compounds. Efforts are underway to use these genes and prote ins directl y in bi oremediatio n of chromate polluted environments.

Natural attenuation Natura l attenuatio n in volves in situ phys ical,

chemi cal and biologica l processes to decrease the concentrati on of a contaminant in the environment over time without human interventio n42

.

Biotransfo rmatio n plays a major ro le in the natural attenuati on of severa l contaminants in lo ng-term contamin ated sites. In a long-te rm tannery waste contamin ated site at the Mount Barker site in South Aus tra li a, industri a l di scharges o f the waste ceased about 25 years ago. Analys is of samples revealed almost same C r(VI) levels in the so il (around 40 mg kg· i

) and wate r (up to 2 mg L·1) at 204 and 2543 years

after the last waste input. Thus, during 5 yea rs ( 1997 -2002), the re was no apprec iabl e natura l attenu ati on o f Cr(VI) at thi s site a lthough the so il was ri ch in organic carbon (9.8-1 5.7%) and harbored C r(V I) reduc ing mi croorgani sms44

. Incubati o n of thi s contam inated so il w ithout and with added cow manure under saturated conditio ns led to complete d isappearance of Cr(VI) within 20 days; but Cr(VI) reappeared probabl y due to reox idatio n of C r(TII) when the sa turated so il was subsequentl y subj ected to drying. However, no decrease in the concentration o f C r(V I) occurred in the M ount Barker so il held at 70% water ho lding capacity even in the presence o f cow manure. Although C r(VI ) can be reduced by a w ide range of aerobic microorgani sms (See Table I), its reducti on in the contaminated so il occurred under saturated conditi ons and not at 70% water ho lding capac ity . Ev idently , reox idati on o f Cr(IlI) and mo isture stress conditio ns woul d probably ex pl ain the lack of natura l attenuati on of Cr(V I) In the contaminated so il at the Mo unt Barke r site.

A wide va ri ety of heterotrophi c mi croorgani sms is in vo lved in the reduc tio n o f C r(V I) to Cr(lll), aerobica ll y o r anaerobica ll y depend ing o n the

organism, both in so il and wate r environments~ 5 . Cr(VI)-tolerant and sensitive bac teria, w ith ability to transform Cr(VI) to C r(lll), occur widely in di verse ecological condition : wate r, sediments and so il 31

.

Ev idence suggests that Cr(VI) reducing microorgani sms are ubiquito us in so il s and can enhance the detox ificati on of C r(V I) under ideal phys ico-chemical conditi ons29

.

Direct Cr(VI) reduction In so il s, mi crobi al C r reducti on may occur direc tl y

or indirectly. In the direc t mode, Cr is taken up by the microbes and then enzy mati ca ll y reduced9

.23

.46, whi le in the indirect mode, produc ts (reducti on or ox idati on) of microbi al decompositio n in the so il such as H2S mediate the reduc tio n o f Cr(VI)30. Di rect microbia l reducti on of C r(VI) was first reported in 1970s24

,47

when certain Pseudomonas strains, iso lated fro m chromate-conta ining sewage sludges, could reduce chromate, dichromate and crocoite duri ng anaerobic growth . Since then, severa l bacteri a with excepti onal ability to reduce Cr(V I) have been isolated from Cr­contaminated and uncontaminated soi l samples. Microorgani sms, impli cated in direct or ind irec t reducti on of Cr(V I), are li sted in T able 1.

Cr(VI) reduction in microbial cultures Since the first reports o f iso lati on of fac ultati ve

anaerobi c Cr(V I)- reducing bacteria in mid- 1970s24,

literature is abundant with instances o f the reducti on o f Cr(VI) by severa l mi croorgani sms, bac teria in parti cul ar48

, mostl y iso lated fro m Cr-impacted environments (T able I ). S tra ins of Oscillatoria, Ch lorella and Zoog /oea have also been repo rted to enzy matica ll y reduce Cr(VI )9. But, as noti ced with bacteri a l res istance to Cr(VI ), Cr(VI)-reducing bacteri a have been isolated a lso from environments w ith minimum or no impact o f Cr29

.49.50. It is a lso

interesting to note that pure cultures of microorgani sms, not previo usly exposed to Cr(V I) , were capable of reducing it49

. Although the exact mechani sm is not known, microorganisms capable of reduc ing Cr(VI) aquired the enzy mes fo r degrad ing re lated compounds present in the enviro nment or produce the reductants that in turn reduce Cr(V l) by chemical redox reac ti ons. Anaerobic chromate reducing strains are prevalent in subsurface so il s and probably enhance Cr reduction in thi s enviro nment2Y

•

Cr(VI)-res istance and reduction are not necessaril y interlinked . Cr(VI) may be reduced by both Cr(VI)­res istant and Cr(V l) -sensi ti ve stra in s of bacteri a and

KAMALUDEEN el al. : BIOREMEDIATION OF CHROMIUM CONTAMINATION 977

not necessaril y all Cr-resistant bacteri a can reduce Cr(VI) . For instance, some aerobic Cr(VI)-resistant bacteri a were not capable of reduc ing itso.sl. Cr(VI) reduction in aerobic conditio ns may not be a resistance mechani sm in bacte ri a, but a "trivia l side activity o f the reductase that may have evolved on other substrates27

. In a bioprocess strategy fo r effecti ve bioremedi ation of Cr(VI), it is important to use Cr(VI)-resistant microbes, w ith ability to reduce it. Two strains o f Pseudomonas jluorescens, o ne resistant and the other sensiti ve to Cr(VI), reduced Cr(VI) at comparable rates20

.48. Likew ise, three Cr(VI)-sensiti ve bacteri a from an uncontaminated soil and three Cr(VI)-res istant bacte ri a fro m two metal­stressed foundry soils and a tannery readil y reduced Cr(VI) anaerobicall / 9

. Inte restingly, Cr(VI)-sensiti ve BaciLLus sp. from the uncontaminated so il was the most effecti ve in reducing C r(VI) among the three Cr(VI)-resistant bacteri al strains from meta l stressed soils and three Cr(VI)-sensiti ve bacterial stra ins from the uncontaminated soil. These bacteria grew aerobically in acetate minimal medium supplemented with sodium chromate, but reduced Cr(Vl) only anaerobically. in the suspension of resting ce ll s of aerobica lly grown bacteri a . Anaerobic growth of the bacterium at the expense of C r(V I) as e lectron acceptor was neglig ible. Con versely, an Arthrobacter sp., iso lated from a long-term tannery waste contaminated soil , was res istant to Cr(VI ) at 100 tLg/mL , but could not reduce it at thi s concentration44

.

Likewi se, Cr(VI) reducti on occurred equa ll y rapidly with both Cr(VI)-res istant and pl asmid-cured Cr(V I)­sensiti ve stra ins of P. jluorescenio. Ch romate resistance determinants have been described o n plasmids in severa l bacteri a, espec iall y in Pseudomonas. But, C r(VI) reduction dete rminants have not been found on pl asmids. Cr(VI) reductio n was independent of chro mate res istance, confe rred by pl asmid pLHB 1, in P. jlourescenio. But, in P. mendocinas2, plasmid pARI 180 determined both chromate res istance and Cr(VI) reducti ons3 .

Microorgani sms, known to reduce Cr(VI), reduce it under ae robic and I or anaerobi c conditions, bu t the phys io logical role in such transformati ons is not c lear. Earli er reports24

.26 have shown that fac ultati ve

anaerobes (Pseudomonas and Aeromonas stra ins) reduce Cr(Vl ) to Cr(lII) anaerobically. A naerobic bacteri a wi th great Cr(VI)-reducing potenti al are ubiquitous in both Cr(VI)-contaminated and uncontaminated so il s29.54

. There is no convincing evidence yet to suggest that Cr(VI) serves as the

e lectron acceptor to support the anaerobic growth of bacteria. E. cloacae grew well under aerobic conditions and slowly under anaerobic conditions at chromate concentrations above 10 mM in nutrient bro th , but could reduce chromate only unde r anaerobic conditio nsso. Al so, there is ev idence that O2

inhibited the reduction of Cr(VI) by Enterobacter cloacae strain HO I in a medium containing other carbon sources as e lectron do nors23.5o. Li kew ise, E. coli could reduce Cr(VI) only in the absence of 0 2SS.S6 . U nder anaerobic conditi ons, Cr(V I) serves as a terminal e lectro n acceptor through e lectron transport systems involving cytochrome c in Enterobacter cloacaeso, cytochro me band d in Escherichia colis7

and cy tochrome c3 in Desulfovibrio vulgaris45.

Membrane or soluble fractions may be in volved in the reduction of Cr(VI). Under aerobi c conditions, both NADH and endogenous cell reserves may serve as e lecron donors for Cr(VI) reduc tio n. A recent stud/8

establi shed a re lati onship between the bioavailab ility of H2 and chromate reductio n in anaerobic aq uifer sediments. The anaerobic enrichment, developed fro m the sediment , utili zed Cr(VI ) and was dependent on H2 for growth and chromate reduc tion. In the absence of Cr(VI ), H2 accumul ated in the anaerobic med ium . But, under Cr(VI) reducing co nditi ons, no H2 and methane accumulated due to utili satio n of the H2 by the enrichment. When H2 was provided in the medium as the e lectron donor the enrichment could reduce 40 mg L-1 C r(VI) in 6 days. Increas ing the ava ilabili ty of H2 by additi on o f suitable e lectron donors (formate, H2 and g lucose) accele rated the reductio n of chromate.

Gram positi ve bacteri a, capable of reducing Cr(Vl ) as a terminal e lectron acceptor and with a relati vely high level o f res is tance to chromate, have been isolated fro m tannery e ffluents II.S9.60 . A chromate­

res istant Gram positi ve bacterium (ATCC 700729) to lerated high concentrations (up to 80 mg/mL ) of d ichromate and reduced 87% of the C r(VI) in 20 mg K2Cr20 7/mL in 72 hr in a nutrient-rich mediu mS'>. T he bacterium could reduce Cr(Vl) even at a concentration of dichro mate as hi gh as 80 mg/mL, but took lo nger time for its reducti on at 80 mg/mL than that required at 20 mg/m L. Ch romate reduction occurs e ither anaerobica ll /0.49.5o.61, aerobica lly17.61,

and . under both conditi ons62 . Agrobacterium radiobacter EPS-91663 and Escherichia coli A TCC 33456 could reduce Cr(VI) under both aerobic and anaerobic conditions. Likewise, a pseudomonad, iso lated from a wood preservation s ite contaminated

978 INDIAN J EXP BIOL, SEPTEMBER 2003

with chromated copper arsenate, reduced chromate under both aerobic and anaerobic conditions62

. Also, Cr(YJ)-tolerant (>400 jkg/mL) facultative anaerobes (five isolates of Aerococcus sp.; two isolates of Micrococcus sp.; and one isolate of Aeromonas sp.), iso lated from tannery effluent, apparently reduced Cr(YI) both anaerobically and aerobicall/,4. Cr(YI) reduction by these facultative anaerobes in diluted peptone water was more pronounced under anaerobic conditi ons (73-94% reduction) than under aerobic conditions ( 18-63% reduction) . But conditions used for anaerobi c and aerobic systems have not been described. Since peptone a lo ne may cata lyse chemical reduction of Cr(YI)6! it was not c lear whether the Cr(YI) reduction in mjcrobial cultures was caused chemically, microbially or both. Cell suspensions of Pseudomonas pUlida PRS 2000, P. jluorescens LB303 and Escherichia coli AC80 aerobicall y reduced Cr(YI) to Cr(1II)27. Reductio n of Cr(YI) in ce ll suspensions of these bacteria was more rapid and complete aerobically than anaerobically . After disruption of the cells of P. pUlida and centrifugation , the supernatant, but f1 0 t the membrane fraction (pellet) , reduced all the added Cr(Yl) within 1 hr. Likewise, Wang and Shen6! reported that resting cells of Bacillus sp. and Pseudomonas fluorescens LB300 aerobically reduced Cr(YJ). But, Cr(YI) reduction by the ce lls of Escherichia coli was inhibited in the presence of oxygen55

.56

. Enlerobacler cloacae, a chromate resistant strain could grow in the presence of Cr(YI) under both aerobic and anaerobic conditions, but Cr(YI) was reduced only anaerobicall /o. The strain lost both res istance and Cr(Y I)-reducing ability on anaerobic growth on nitrate.

Cifuentes el al .32 reported that organic amendments enhanced the reduction of Cr in soil s by indigenous microflora. Generally , Cr(YI) reduction by growing bacterial cells has been demonstrated in media containing natural aliphatic compounds, amino acids and fa tty ac ids as e lec tron donors6!. Microbi al reduction of Cr(YI) occurred during anaerobic degradation of benzoate4o. Diss imil atory meta l­reducing bacter ium, Shewanella oll eidensis could reduce Cr(Vl) when grown on fumarate or nitrate as an electron acceptor and lactate as an e lectron dono/'s. Cr(Y I) reduction under fumarate and deni tri fyi ng condit ions, dependent on the physiologi cal sta te of the c ulture, ,vas possibly inducible under anaerobic cond itions. Cr(VI) reduct ion in the anaerob ica ll y grown stationary phase

of thi s bacterium is a complex process, poss ibly involving more than one pathwa/ 6.

A wide range of organic pollutants such as phenol , 2-chlorophenol, p-cresol, 2 ,6-dimethylphenol, 3,5-dimethylphenol , 3,4-dimethylphenol, benzene and toluene can also serve as e lectron donors for Cr(VI) reduction in cocultures contallllllg E. coli A TCC33456 and P. pUlida DMP_1 4o. Metabolites produced during phenol degradation by P. pulida served as e lectron donors for Cr(VI) reduction by E. coli. Technology using such cocultures would help to simultaneously detoxify both organic pollutants and the toxi c Cr(VI).

Non-metabolising resting cells of bacteri a could reduce Cr(VI) , but only in the presence of an added carbon source20.56,67 . Killed resting cells could not cause Cr(VI) reduction56

.6!. Soluble enzymes in cell

extracts can reduce Cr(VI) in the presence22. 67 or

absence2o. 56 of added electron do nors.

According to very recent evidence, nonmetabolic Cr(VI) reduction can occur on bacteri al surfaces even in the absence of ex ternally added electron donors in the meoium. Thus, Fein el al.68 demonstrated that non metabolizing cell s of Bacillus subtilis, Sporosarcina ureae and Shewanella putrefaciens could reduce significant amounts of Cr(VI) in the absence of externally supplied e lectro n donors. The Cr(VI) reduction by the bacterial strains was dependent on solution pH, decreasing with increasing pH, and presumably occurred at the cell wall and independent of the oxidation of bacteri al organic exudates. Such non metabolizing reduction of Cr(VI) by bacteria in nutrient-poor conditions may be important in the biogeochemical di stribution Cr.

Cr(VI) reduction by microorgan isms, known to occur under both aerobic and anaerobic conditions (see Table 1), is a redox-sensitive process56

.69

. The ability of washed resting cells of Agrobacterium radiobacter EPS-916 to reduce Cr(VI) was governed by their redox potenial63 . Resting cells of A. radiobacter EPS-916, pregrown under aerobic conditions on g lucose, fructose, maltose, lactose, mannito l or glycerol as the sole carbon and energy source, ex hibited similar redox potentia ls o f around -200 mV and completely reduced 0 .5 mM chromate. On the other hand, the inability of the resting cells of the bacterium, pregrovm on g lutamate or s ll ccinate, to red uce chromate was associated with relatively hig h redox potentials of - 138 to - 132 m V. Moreover, rest ing cells, pregrown under anaerobic conditions on glucose, had lower redox potentia ls (-240 mY) and a

KAMALUDEEN et at.: BIOREMEDIATION OF CHROMIUM CONTAMINATION 979

more pronounced chromate-reducing activity than did the aerobically grown resting cells on glucose with redox potenti al of -200 mY . Likewise, cells, pregrown anaerobically on chromate as the e lectron acceptor, effected more rapid reduction of chromate than did the anaeorobically grown cells ( -198 mY) on nitrate. Evidence suggested a negative corre lati on between chromate reduction by the resting cells of A. radiobacter EPS-9\ 6 and their redox potential. But, on the other hand, in an anaerobic enri chment from aquifer sediment, Cr(YI) reducti on appears to occ ur under nitrate reducing conditions, but before iron and sulfate reduction58. Evidently, hi ghly reducing conditions, necessary for the reduction o f iron and sulfate and methanogenesis, may not be required for chromate reduction. However, it was observed that there existed a competitIOn among chromium reduction and nitrogen reductio n. Clostridium and Methanosarcina reduced both nitrogen and chromium. In the presence of chromium nitrogen reduction was reduced 70. Actual mechani sm of competition needs verification.

Abiotic reduction of Cr(YJ) has also been demonstrated in media rich in nutrients containing some reductan ts, espec ially under predominantl y reduced conditions. Thus, even in sterile tryptic soy broth, Cr(Yl) was reduced abiotically with time as a function of redox potential 29 . Thus, more than 50% of the 25 Ilg Cr(YI)/mL added to the tryptic sterile broth was reduced abiotically in 60-80 hr at redox potenti a ls of -120 and -380 mY , as compared to <27 % reduction at +243, +1 86 and +58 mY during the corresponding peri od. It is therefore necessary to have appropri ate control to exclude the chemical redox reaction s when nutrient-rich growth media are used to assess the Cr(VI)-reducing ab ility of pure cultures of

. . microorgalllsms.

Cr(VI) reductases Cr(Y I) reduction is mediated enzymatically (direct)

and/or non-enzy matically (indirect). There IS

considerable literature on the in voivement of Cr(YI) reductases in direct reduction of C r(Yl) to Cr(IIJ) by bacteri a. In grow ing cultures with added carbon sources as e lectron donors and in ce ll suspensions, Cr(YI) redu ction can be predominantly aerobic or anaerobic, but general I y not both . I nteresti r.g l y, Cr(YI) reductases can catalyse reduction of C r(YI) to Cr(l II) anaerobicall/ 5

, aerobica ll y27.71 and also both anaerobically and aerob icall / o.57.62.72 . The Cr(YI)­

reductase enzyme may be present in the membrane

fraction of the cells as in Pseudomonas fluorescens and Enterobacter cloacae28 or in the soluble fraction of the cells (ce ll -free system) as in P. ambigua22

, P. putida27 and a Bacillus Sp.72, with NADH , NADPH or H2 (Desulfovibrio vulgaris) as e lec tron donors and poss ible involvement of cy tochromes b, c and d. Membrane vesicles of E. cloacae, reduced with NADH and then exposed to Cr(YI), ox idi zed c and b cytochromes and reduced Cr(YI). Ev idence suggested tha t specificall y cytochrome c548 was invol ved in the reduction of Cr(YI) by me mbrane ves ic les73

. In the presence of H2 and excess of hydrogenase, cytochrome c3, a peripl asm ic protein , in the soluble cell-free fraction of the ce ll s in D . vulgaris45 reduced Cr(YI), 50 times faster than did the Cr(Yl) red ucta~e

of P. ambigua with NADH and NADPH as e lectron donor22. Soluble fractions of the cell-free ex tract, largely cytoplasmic, of a pseudomonad from a wood preservation site reduced chromate, added at 10 mg Cr(YI)L-\, under both aerobic (55%) and anaerobic (80%) conditions in 2 .5 hr62 . Cr(YJ) reductase in anaerobically grown Shewanella putrefaciens MR-l was formate-dependent with highest activity in cytoplasmjc membrane74 . The Cr(YI) reductase in P. ambigua7 1 and a Bacillus Sp.72 have been puri fied and characterised. More recently , to clone a chromate reductase gene, a novel soluble chromate reductase of P. putida has been first purified to homogeneity and characterized, using ammonium sulfate precipitation, anion-exchange chromatography, chromatofocusing and gel filtration75. The reductase activity was NADH- or NADPH-dependent. The optimu m conditions for the chromate reductase were: 80°C and pH 5.0. Kinetic properti es of the enzy me showed Km of 374 IlM CrO/ - and Ymax of 1.72 Ilmollmin/g of prote in. Suzuki el al.7 1 sequenced the gene encoding the chromate reductase71 fro m P. ambigua. But, the genes encoding the chromate reductase in P. ambigua and P. pUlida were not homologous. A bacteri a l reduction of chromate by a flavin reductase with flavin is known 76 . Furthermore the end product was a soluble stab le Cr(lII)-NAD complex instead of Cr(l Il) precipitate . Since intracellularly formed CrO ll ) forms adduct product with DNA, protein , glutathione, and ascorbate in eukaryotic ce ll s, the reported bacteri a l flavin dependent reductase in bacteria will protect the ce ll s

Reduction products Generally , in bacteri al cultures or in enzyme

systems, Cr(YI) is reduced to Cr(lll) withou t

980 INDIAN J EXP BIOL, SEPTEMB ER 2003

transitory accumulatio n o f any intermediate. But, there are in stances when Cr(V) accumulates as a transitory intermediate during microbia l convers io n o f Cr(V I) to Cr(IlI). For instance, the NADPH­dependent Cr(VI) reductase in P. ambigua ca ta lysed the transitory fo rmation o f C r(V) during convers io n of Cr(VI) to Cr(1lI )7 1. Tox ic ity of Cr(VI) to microorganisms is pro bably associated with the transient fo rmation of Cr(V) as an intermedi ate. Cr(V) is fo rmed also during reductio n of Cr(VI) in alga l cultures and in reactions with phys io logical reducing agents such as NA DPH , g lutathi one and severa l pentoses 77 .

In most studies, conclus ions o n mi crobia l reductio n of Cr(VI) were based on its di sappearance ancl/or accumulation of the Cr(IlI) [determined as the difference in total Cr and Cr(VI)] as the reduction product with incubatio n. The colorimetric dipheny lcarbazide method commo nly used in Cr( VI) es timation is not specific since its probable reduction product Cr(V) and hexavalent forms o f Mo, V and Hg can a lso react w ith the same reagent. But, the direct measurement of the oxidation state of the Cr during bacte ri al reducti on of C r(VI ) has not been attempted until recently . Daulton el al.78 used the electron energy loss spectroscopy (EELS) technique to characteri ze the oxidation state of C r during Cr(VI) reductio n by Shewanella oneidensis in anaerobic cultures. TEM of the cells exposed to Cr(VI) sho wed that the cells were encrusted in Cr-ri ch precipitates, mostly res tricted to the o ute r surface of the ce ll s. T hese prec ipitates, based on analysis by EELS , contained Cr(Ill) or its lower state o f ox idati on. Myers el al. 74

, using e lectron paramagnetic resonance (EPR) spectroscopy, confirmed the fo rmati on of Cr( V) via one-e lectron reducti on o f C r(VJ) as the first s tep by a facultati ve anaerobe Shewanella pUlrefa ciens MR-I .

Indirect reduction Apart from the direct (enzy matic) reducti on of

Cr(VI), microorganisms can also medi ate the reducti on o f Cr(VI) ind irectl y, invo lving a bioti c­abiotic coupl ing. For instance, Fe(ll ) and S2., prod uced by microorgani sms th ro ugh di ssimil atory reductio n path ways, can chemically catalyse several biogeoche mi ca l processes inc luding Cr(VI ) reduc ti on 79.80. Fe(I IJ), an important e lectron acceptor fo r microbi a l ox idation of organic compounds (a li phatic and aromatic) , is o ne of the most abundant metals in the soil. A wide range of bacteri a couple the

oxidation of organic compounds and H2 to reductio n of Fe(III) and S04 to Fe(II) and H2S, respective ly under oxygen-stress conditions8o. Thi s occurs as in submerged rice soils for example . Diffe rent genera of Fe(III)-reducing bacteria reduce Fe(JU) via different mechani sms81. Recently , Wielinga et al. 82 demon­strated the reduction o f Cr(VI) by a bio ti c-abio ti c coupling mechanism invo lving iron reduction. Diss i mi latory Fe(Ill) reduction by Shewanella alga ATCC 511 8 1, a facultative anaerobi c bacterium, under iron reducing conditions prov ided a primary pathway for chemjcal reduction of C r(VI) , injec ted into a bioreactor, by microbi a lly induced ferrous ion. However, it has been diffi cult to d iffe renti ate the exact contribution between bio logical (direc t) and che mical (indirect: bi oti c-abiotic) reduction of C r(VI) in a soil environment. Evidence using Desulfovibrio vulgaris · as a model chromate reducer suggests that chemical reducti on of chromate by Fe( II) was 100 times faste r than that by D. vulgaris, a chromate reducer82 . In anaerobic environments abundant in Fe(Il), no nenzy matic reduction of Cr(V I) by Fe( lI ) can be as important as enzymatic Cr(V I) reduction83. A facultative anaerobe Pantoea agglomerans SP L

coupled anaerobic growth on acetate and other e lectron do nors to the diss imil atory reduc tion of e lectron acceptors, Fe(III), Mn(IV) and Cr(VI), but

not sulfate84 . When Cr(VI) was added to thi s y­protobacterium culture with e lementa l sulfur alo ne, SO-di sproportionation to sulfate and hydrogen sulfide occurred with concomitant grow th of the bacterium and reduction of Cr(VI)85. Likewise, P. agglomerans SP 1 grew chemo lithoautotrophi ca lly by the SO­di sproporti onati on, co upled to reducti on o f Fe(III ) and Mn(lV). Probably, SO-di sproportio nation that may be widespread in certain anaerobic environments may prov ide an effecti ve mechani sm for attenuati on of C r(VI) through its reductive detox ification.

Sui fate-reducing bac teri a (obligate anaerobic hete rotrophs) couple the oxidation of organic sources to the reducti on of sulfate to sulfide. Reducti on of C r(VJ ) by bacteri ally produced hydrogen sul fide, fo llowed by prec ipitation of the Cr(lll) formed, is an important mechanism in sul fa te-rich so il environ-

h b · d " '1318687' ments w en an aero IC con Itlo ns preval . . , as In

fl ooded compacted so il s. Likewise, sulfide, produced by sulfate- reducing bacteria , has been implicated in C r(VI) reducti on in marine environments88 . Hydrogen sulfide, produced in ac id sulfate soil under reducing conditions, is eas il y precipitated as FeS in reduced soils89 and sediments. Fe(II )90 and H2S87 , both

+

KAMALUDEEN et 01.: BIOREMEDIATION OF CHROMIUM CONTAMINATION 98 1

microbially produced, are effecti ve reductants of Cr(VI) under reduced conditions as is the FeS91

. Low concentrations of Cr(Vl) can accelerate the growth and sulfate-reducing acti vity of sulfate-reducing bacteri a92 and thereby the reducti on Cr(VI) by the H2S evolved. Interestingly, a spore-forming sul fa te­reducing bacterium, Desulfotomaculum reducens sp. nov . strain MI-l , isolated from sediments with hi gh concentrations of Cr and other heavy metals by enrichment, could grow with C r(Vl) as sole e lectron acceptor in the absence of sulfate with butyrate, lactate or vale rate as the e lectron donor9J . Cr(VI) was presumably reduced to C r(lII) as C r(OH h In the absence of Cr(V I), no bacteria l growth was noticed .

Biologicall y generated sulfur compounds with high reducing power such as sul fite , thiosul fa te and polythionate can cata lyse the chemical reducti on of Cr(VI). Chemoautotrophic ba~teria , belonging to Thiobac illi group, that can deri ve energy fro m the oxidation of inorganic sul fur compounds during sulfur oxidation , generate a range of sul fur compounds such as sul fite and thiosulfate with high reducing power that can in turn catalyze the reduction of Cr(VI). For instance, Thiobacillus fe rroxidans, grown on elemental sul fur, has been used to reduce Cr(VI) under aerobic conditions94

.95 . The Cr(VJ)-reducing

ability of the cells of this bacterium under aerobic conditi ons in shake fl asks and in fe rmentati on vesse ls was related to the generatio n of protons with high reducing power fro m elemental sul fur96

. T. ferroxidans could reduce Cr(VI ) over a wide pH range (2-8), interesting ly with more pronounced reduction at lower pH, associated with increased ox idati on of e lemental sulfu r to products with high reducing power. Cr(VI) reduction, medi ated by T. ferroxidans in the presence of e lemental sul fur, occurred under both aerobic and anaerobi c conditions, but more effecti vely under aerobi c conditio ns. Ev idently, bacteri a l reduction of C r(VI) , in volving bioti c-abiotic coupling , can occur under both sul fa te­reducing and sul fur-ox idi sing conditio ns. Thus, Cr(VI) reduct ion or immobili sation can be effected ab ioti ca ll y by diffe rent substances; but, there is considerable progress in recent years o n the feas ib ility of using bio logical reduction for treatment of Cr(V I)­containing was tes .

Several remediati on techn iques are ava il ab le fo r so ils contami nated with C r. Techno logy applicable to a particular Cr site depend o n the c lean up goals, the form of Cr present and vo lume and phys ico-chemi cal conditions of the contaminated environments like soil ,

water and sediments. Processes, developed for remediation of environments contaminated with chrome wastes, are more suited for aquatic systems than for terrestri al systems. Traditional methods, used especially for waste waters, involve c hemical or e lectrochemical reduction of Cr(VI) to Cr(lll ), precipitation of the latter and its removal by fil tration or sedimentation9o. Chemical methods are generall y not cost effecti ve and may themselves generate hazardous byproducts79 . Microorgani sms are capable of a ltering the redox state of Cr by reducing Cr(VI ) to C r(lll) through direct (enzy matic) or indirect (v ia iron reduction, sul fa te / sul fur reductio n or sul fur ox idation) processes .

Bioremediation technologies for waste water/solution

Biosorption Sequestration and immobilisation of heavy metals,

especially in the solutions of effluents and waste water, can be accompli shed th rough biosorption, a passive process of metal uptake, using biomass (dead bio mass in parti cular)97. Biosorption is essentially a non-directed phys ico-chemical complexation reaction between di ssolved metal species and charged cellular compo nents, that involves sorption and/or complex ing o f metals to li ving or dead cells. The precipitation or crystalli sation of metals leading to their sequestration can take place at or near the cell. Al so, insoluble metal species can be physically entrapped in the microbi ally produced ex trace llular matri x or precipitated 111 bacteri a l or algal exudates98 . Extracellular matrices may consist of neutral po lysaccharides, uronic ac ids, hexosamines and organica lly bo und phosphates that are capable of complex ing metal ions. Metabo licall y medi ated accumulation is usuall y intracellul ar and linked to the contro l of plasmid linked genes99

.

Yeasts and bacteri a as well as algae can effecti vely sequester metals in solu tions 1OO

, because of their metal-binding capabi lities. Algae, such as Scenedesmus, Selenastrum and Chiarella, are known to bioaccumul ate metals 10 1

. The functional groups present in the cell s and cell walls of fun gi and algae can serve as the probable sites for biosorption of metals. For instance, the amino group of chitin (R2-NH) in alga Sargassam and chitosan (R- NH2) in fungi are probabl y the effecti ve binding site for C r(VI) . But, functio nal groups such as chitin and chitosan seem to contribute only 10% of the metals sequestered by the biomass.

982 INDIAN J EXP BIOL, SEPTEMB ER 2003

Biosorption , using especially dead biomass, is a cost-effective technology for removal of heavy metals, and is as effective as ion exchange, but is yet to be exploited commercially. Biosorption research was confined to mostly cations and there is a need for research on uptake of anions by biomass such as Cr. Biosorption of Cr(VI) is often followed by its bioreduction to less toxic Cr(III ) and eventual precipitation of the latter. Bioreduction has been used for removal o f Cr(VI) from wastewater systems in METEX anaerobic s ludge Oreactor, BIO-S UBSTRAT anaerobic micro-carrier reactor and Agarkar Research Institute chromate reduction process 102 . Biosorption is not suitable for detoxification of solid Cr-wastes in so il s.

Biofllms in bioreactors Bacterial bio films have been recommended as an

efficient means of remediating contaminants in the environment, because, biofilms provide tolerance to des iccation , a high level of pollutants and other stress factors. Smith and Gadd ,03 used a mixed culture sulfate reducing bacteri al film for reduction of hexavalent Cr. In the presence of lactate as the carbon source and sulfate, 88% of the 500 /lmol of Cr(VI) was removed from the solution with bacterial biofilm as insoluble Cr(lll) in 6 hr. Since sulfide, a reductant o f Cr(VI), was not detected in the medium and no reduction occurred in uninoculated medium, di ss imilatory chemical reduction was not involved in Cr(VI) reduction. Evidentl y, Cr(VI) reduction in sulfate reducing bacterial films was biologically mediated, presumably by enzymes. It is also possible to recover the insoluble or precipitated Cr(I1l) from the bacterial films. There is scope for using thi s biofilm technology for detoxification of Cr wastes in a bioreactor.

lmmobilised cells Ce ll s immobili sed on polyacry lamide ge l can be

used for effective detoxification and removal of metals in solution from e ffluents in a reac tor. Intact ce ll s of a sulfate reducing bacterium Desulfo vibrio desuljilricans, immobili sed o n polyac ry lamide ge l, reduced around 80% of 0 .5 M Cr(VI) with lactate or H2 as the electron donor and Cr(Vl) as the electron acceptor l04

. Insoluble Cr(IlI) accumulated on the surface or interior of the gel. Immobilised cells also effected the reduction of other ox idised metals, Mo( VI), Se(VI) and U(V!). lmmobilised ce ll s may be useful fo r detoxifi cation of Cr(VI) in bioreactors.

Bioreactor using living microorganisms Rajwade and Paknikar ,05 devel oped an efficient

chromate reduction process using a strain of Pseudomonas mendocina MCM B- 180 for treatment of chromate-containing wastewater. The bacte ri al strain used was resistant to 1600 mg Cr(VI)L" and reduced 2 rnM chromate [100 mg Cr(Vl) L·I] in 24 hr. In 20-mL continuously stirred bio reactors containing this bacterium and sugarcane molasses as a nutrient, 25-100 mg chromate L'I was removed within 8 hr52

" o2. Efficiency of this bioremediation process is enhanced by anaerobiosis.

Conclusion Compared to other conventional methods,

bioremediation is highly economical and ecofriendly as thi s generates no furth er waste into the environment. Challenges till re main in the form of elevated concentrations of Cr(VI) in groundwater and in deeper soil profiles that needs further research in the field of bioremediation.

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