Indian Journal of Experimental Biology Vol. 41 , September 2003, pp. 935-944

Microbes in heavy metal remediation

P Rajendran, J Muthukrishnan & P Gunasekaran*

Department of Microbial Technology, Madurai Kamraj University , Madurai 62521, India

Heavy metal contamination due to natural and anthropogenic sources is a global environmental concern. Release of heavy metal without proper treatment poses a significant threat to public health because of its persistence, biomagnification and accu mulation in food chain. Non- biodegradability and sludge production are the two major constraints of metal treatment. Microbial metal bioremediation is an efficient strategy due to its low cost, high efficiency and ecofriendly nature. Recent advances have been made in understanding metal - microbe interaction and their application for metal accumulation/detoxification. This article summarizes the potentials of microbes in metal remedi ation .

Keywords: Bioremediation techniques, Heavy metal contamination, Metal bioremediation, Microbial metal bioremediation .

Contamination of heavy metals in the environment is a major global concern because of their toxicity and threat to human life and environment l-2 . Much research has been conducted on heavy metal contamination in soils from various anthropogenic sources such as industrial wastes3, automobile ernissions4, mining activit/, and agricultural practices6. The group of heavy metals are about 65 and are defined in a number of criteria such as their cationic-hydroxide formation , specific gravity greater than 5 glml, complex formation, hard-soft acids and bases, and, more recently , aSSOCiatIOn with eutrophication and environmental toxicity .

Roane and Pepper7 classified metals into three classes on the basis of their biological functions and effects: (1) the essential metals with known biological functions, (Na, K, Mg, Ca, V, Mn, Fe, Co, Ni, Cu, Zn, Mo, and W), (2) the toxic metals (Ag, Cd, Sn, Au, Hg, Ti , Pb, AI , and metalloids Ge, As, Sb and Se), and (3) the nonessential , nontoxic metals with no known biological effects (Rb, Cs, Sr, and T). Based on primary accumulation mechanisms in sediments, heavy metals are classified into five categories. (I) adsorptive and exchangeable, (2) bound to carbonate phases, (3) bound to reducible phases (Fe and Mn oxides), (4) bound to organic matter and su lfides, and (5) detrital or lattice metals8.

Need for metal bioremediation Greater awareness of the ecological effects of toxic

metals and their biomagnifications through food chain

*Correspondenl aUlhor : Phone: 0452-5456478; 452-458209 Fax: 0452-5458478; 0452-459139 Emai l pguna@elh.nel

as well as highly publicized episodes such as mercury pollution in Minimata, Japan has prompted a demand for decontamination of heavy metals in the aquatic systems. However, essential metals are required for enzyme catalysis, nutrient transport, protein structure, charge neutraliziation and control of osmatic pressure9. Olson et al. ID reported incorporation of nickel in four microbial enzymes involved in ureolysis, hydrogen metabolism, methane biogenesis and acetogenesis . A number of heavy metals are required as rnicronutrients to plants. They act as cofactors as part of prosthetic groups of enzymes which are involved in a wide variety of metabolic pathways. However, when they are present in high levels , most heavy metals are toxic to plants I' .

Metal concentration has been linked to birth defects, cancer, skin lesions, retardation leading to disabilities, liver and kidney damage and a host of other maladies' 3. The Center for Disease Control (CDC)'2 and the Agency for Toxic Substances and Disease Registry (ASTDR)'2 estimate that 15-20 % of U.S. children have lead levels greater than 15 mgldl in blood, which is considered potentially toxic . Changes in trace element profile of the soil causes physiological and genetic changes in various life, such as plants, aquatic and benthic fauna, insects, earthworms, fish , birds and mammals as evidenced by recent research work4. Tyagi et al.3 stated that the concentration of heavy metals in industrial areas in India is much higher than the permissible limit of World Health Organization . They have also reported that these metals in the ground water have caused various di seases in human beings and also disturb the metabolic functions. A brief li st of hea,vy metal s with

936 INDIAN J EXP BIOL, SEPTEMBER 2003

their pathological effects, which pose health hazard to envi ronment (Table I ) 14.

Metal availability

Sposito' 5 categorised metals as bioavailable (precipitated, non-sorbed and mobile) and non-bioavailable (precipitated , sorbed , and non-mobile) metals. In nature , metals and Il!etalloids ex ist mostly as catio ns, oxyanions, or both in aqueolls soluti on and mostly as salts or oxides in crystalline (minera l) form or as amorphous precipitates in soluble form 7. ' 6 . The mobility of metals as hydrated ionic salts is dependent on two factors 1. The meta llic e lement that is parti c ipating as positi vely charged io ns (cations) and 2. The one, which makes up negati vely charged component o f the salt. The cationic/anionic solubility re lationship is in T able 2 . Geochemical forms o f heavy metals in soil affect the ir so lubility , which direct ly influence the ir bioavail abili ty '7. Lena and Rao l8 reported mobility and bioava il ability of metals in so il s is in the order of Zn > Cu > Cd > Ni , based on their solubility and geochemical forms. The fa te of tox ic meta ls in so ils depends mainly on the initi al chemical form o f the metal even though the environmental and edaphic conditions such as pH, redox status, and so il o rganic matte r content have signi ficant influence ' '! . Studi es of Sauerbeck and Rietz20 have indicated the ex istence of different binding fo rms and strong ly p H depenent solubility effec ts of the trace e lements. Soil cation exchange capacity (CEC) affect meta l bioavail ibilty and it depends on organic matter and cl ay content o f the so il. The tox ic ity of metals within soils with high CEC is low even at hi gh to tal meta l concentrati on and in contrast the toxic ity is vice-versa in low CEC 7 . Under ox idi zed or aerobic cond iti on (+ 800 to a mY) "metals are usua lly fo und as so luble cati oni c fo rms

(Cu2+, Cd2+, Pb2+ & Ca2+) and in reduced or anaerobic conditions (0 to -400 mv) they are fo und as CuS , PbS , and CdC03, precipitates

7. At low soil pH, the

meta l bioavailability increases due to its free ionic species and in contrast, high so il pH decreases due to insoluble metal minera l phosphate and carbonate formation . However, in contrast to other metal bioavailabilities, at high pH range o f 5-9, nickel may be adsorbed on iron and manganese ox ides, or form complexes with inorganic ligands (OH-, S042- , cr or NH3)21.

Metal microbe interaction Microbial interactions with heavy meta ls were

reviewed by, Ehrlich ' 6 and Beveridge and Doy le22 . Microbial interactio ns with small quantiti es of meta ls or metalloids do not exert a maj or impact on metal or metalloid d istribution in the environment, whereas interactions with larger quantities are required in energy metaboli sm, fo r instance, to have noticeable impac t ' 6. However, due to strong ionic nature metals

Table 2 - Summary of cati on/ani on solubi li lY relationshi p of metals

Anions Cations Solubil ity

All ani ons Na+, K+, NH/ Solub le

NO ;- , NO -2 ' C2H30 2, All metals Soluble

Mn0 4, CIO ~ , CIO ~

CI- , Br- , 1- All metals except Soluble Pb2+, Ag+, Hg2+

SO ~ - All metals except Soluble Ba2+, Sr2+, Pb2+

0 2-, S2-, OW All metals except Insoluble Ca2+, Ba2+, Sr2+

CO ~ - , PO ~- , SO ~- All metalli c salts except Inso luble

BO j- , F , SiO j-Na+, K+, NH/

Metal Use

Table I - Applicati on of heavy metals and their tox ic effects

Effect

Hg Coal. vinyl chl orides. electri ca l balleries

Pb

A I'

Cd

Cr

Zn

Co

Se

Ni

Bc

Pl as tic, paint, pipe, balleries, gasoline, auto exhaust

Pes ti cides, coal. detergents

Ferti I ize r, pl astic, pigment

Tanning, paints, pigmenl , fungicide

Fert il izer

Vilamin B- J2

Coa l, sulfur

Electroplating

Coal, rocket fuel

Neurotoxic

Livercirrohosis, meni al disturbance, cancer, ulcer and hypokerotos is

Kidney damage, injury in cns and mental retardation

Nephritic, cancer and ulceration

Vom iting, renal damage and cra mps

Diarrhoea. low blood pressure and paralysis

Damage of li ver, kidney , spl een and nervousness

Teratogeni c, carcinogenic, genotox ic and mu tagen

Carcinogen, acute and chron ic poison

RAJENDRAN e( al.: MICROBES IN HEA VY METAL REMEDIATION 937

bind to many cellular ligands and di splace nati ve essential metals fro m the ir normal binding sites, which are tox ic7. Eubacteri a and archaea are able to

ox idize Mn (II), Fe (II), Co (Ill), AsO ; , Sea or reduce

Mn (IV), Fe (Ill) Co (II) AsO ~ - , SeO ~ on a large

scale and conserve energy in these reac ti ons '7. So me microbes reduce ions such as Hg2+ or Ag+ to HgO and AgO respec ti vely, but do not conserve energy fro m these reacti on23 . Prokaryotes methy late meta l and meta lloid compounds producing corresponding volatil e metal deri vati ves24 . The bacteri a l ox idati on o f

AsO ; to AsO !- by a stra in o f Alcaligenes faeca lis

and reduction of CrO ;- to Cr (OHh by Pseudomonas

j luorescens LB 300 or Enterobacter docae are examples of redox reactions involving enzymati c microbi al detoxification of harmful meta ls or metallo ids25 . Microbes secrete inorganic metabo lic products such as sulfide, carbonate o r phosphate ions in the ir respiratory metaboli sm and with them precipitate tox ic metal ions as a form of non-enzymatic detoxification26. In nature mi crobes immobili ze metals through cellul ar sequestration and accumulation, or through extracelliular

.. . '7 preclpltatJOn- .

Conventional method of heavy metal removal from soil and water

Various methods are available fo r the removal and management of heavy meta ls, which invo lve technical inputs. Current methods used for treating so il s contaminated with tox ic meta l are 1) Land filling: the excavation, transport and deposition o f co ntaminated soil in a permitted hazardous was te land, 2) Fi xati on: the chemica l process ing of soil to immobili ze the metals, usually fo llowed by treatment o f the so il surface to e liminate penetration by wate r and 3) Leaching: using ac id soluti ons as proprietary leaching agent to desorb and leach meta ls fro m so il fo llowed by the return of clean so il res idue to site '9.

Approaches to reduce heavy metal contamin ation in water include: 1) prec ipitati on or fl occul ation, followed by sedimentati on and di sposal of the resulting sludge, 2) ion exchange, 3) reverse osmosis, 4) microfiltration, 5) electrodialys is and 6) evaporation . Apart from these, o ther methods like foa m floatation, liquid membrane technique, solvent extraction and crysta lli zatio n can a lso be employed .

Bioremediation techniques The goal of microbi al remediation of heavy meta l

contaminated so ils and sediments are to immobili ze

the metal in situ to reduce metal bioava ilability and mobility or to remove the metal from the so il. Pazirandeh et al.28 described the metabolic pathways resulting in bioprecepitation o f heavy metals or the ir bio transformation. Low cost and higher effic iency at low meta l concentrations make biotechno logical process very attracti ve in comparison to physico-chemical methods fo r heavy meta l removal29 . Metal remedi ation strategies using mi croorganisms can minimize the bioavail ability and bio tox ic ity of heavy metals3o.31 . Biostimulation, stimulation of viable nati ve microbi al popul atio n, bioaugumentati on, artif ic ial introducti on o f viable populatio n, bioaccumul ation use o f live cells, and bi osorption, use of dead microbi al bio mass, are the cost-effec ti ve innova ti ve bioremedi ation techno logies. Biological approach fo r meta l detox ification affords the potenti al fo r se lective removal of tox ic meta ls and operatio n fl ex ibility and easy adaptability for in situ and ex situ application in a range o f bioreactor confi gurati ons3o. In the past few decades, new meta l treatment and recovery techniq ues based on biosorptio n have been explored using both dead and living microbi al bi omass with remarkable e ffi c iency. Prokaryotic and eukaryoti c microbes are capable of accumulating metals by binding them as cations to the ce ll surface in a pass ive process22 .

Microbes for metal remediation The mechani sms by which metal ions bind to the

ce ll surface include e lectrostati c interac tio ns, Van der Waals forces, covalent bonding, redox interacti ons, and ex tracellular precipitati on, or combinati on o f these processes32 .The negative ly charged groups (carboxyl, hydroxy l, and phosphoryl) o f the bacteri al cell wall adsorb meta l catio ns, which are then retained by mineral nucleatio n33. Biosorption studi es o f U, Zn, Pb, Cd, Ni , Cu, Hg , Th , Zn , Cs, Au, Ag, Sn and Mn, showed that the ex tent o f so rptio n varies markedly with the metal as we ll as with the microorgani sms (Table 3).

Surfactants such as rhamnolipid produced by P. aeruginosa show specific ity for certain meta ls uch as Cd and Pb. Hi gher molecul ar weight (- 106) bioemulsifiers such emul san, can also aid in metal removal35. Studies of Sand et a f. 36 revealed that ThiobacilUus f erroxidans and Leptospirillum fe rroxidans are capable of ox idiz ing iron and sulfur. Joerger et al. 37 reported that metal accumulating bacterium Pseudomonas stutzeri AG 259 is capab le of producing sil ver based single crystals, which can reduce the toxic ity of meta ls.

938 INDI AN J EXP BIO l , SEPTEMB ER 2003

Mechanisms of metal tolerance Organisms respond to heavy metal stress using

di fferent defense systems (Fig. 1) such as exclus io n, compartmenta lization, fo rmation of complexes and synthesis of binding proteins like metallothioneins (MTs) and phytochelatin s (PCs). Ochari 38 has di vided general tox icity mechani sm for metal ions into three categories, 1. blocking the essenti a l biological funct ional groups o f biomolecul es especia lly prote ins and enzy mes, 2. di spl ac ing the essenti al metal ion in bio molecules and 3. modifying the acti ve conformation of biomolecules resul ting the loss of

Table 3 - Example of microorgani sms that take up heavy metals

Microorgan isms

Zooglea spp.

Citrobacter spp

Bacillus spp

Chlorella vulgaris Rhizopus arrh iZ. lIs

Aspergillus niger

Elemen ts

Co Ni Cd U

Cu Zn Au P

Ag Hg Th

Protein denaturation (Hg, Pb & Cd).

Uptake (% dry wt)

25 13 170 900 15 14 10 10 54 58 19

specific actI vIty. Microorgani sms can affec t heavy metal concentrations in the environment because they exhibit a strong ability fo r metal removal from solution ; thi s can be achieved th rough either enzymatic or non-enzymatic mechanjsms39 . A voidance, restriction of metal entry into the cell, e ither by reduced uptake/acti ve efflu x or by the fo rmation of complexes outside the ce ll and sequestrati on, reduction of free ions in the cy toso l e ither by synthes is of ligands to achieve intrace llu lar chelation or by compatmenta li zati on are the two major strategies o f organi sms to protect themselves against heavy metal tox icit/o. The general mechani sms of metal tolerance in microbes are shown in Table 4.

Metallothioneine Metallothi oneines, di scovered about 45 years ago,

playa centra l ro le in heavy metal metabo li sm and in the management of various forms of stress. Meta llothio neines (MTs) are low molecular weight (6-7 kDa), cystine- ri ch prote ins, di vided into three di ffe rent cl asses on the bas is of their cystine content and stru~ture . The Cys-Cys, Cys-X-Cys and Cys-X-X-Cys, motifs ( in which X denotes any amino acid) are characteristi c and invari ant for metallothioneine41 . Studies of Lichtlen and Schaffner42 show metal

Inhibition of cell division (Pb. Cd. Hg & Ni)

/ MIC ROB ES

uption Cell membra ne disr (Hg, Pb. Zn. Ni. Cu &Cd)

Inhibiti (Hg.

on of enzyme activity ] Pb. Zn, Ni. Cu & Cd)

Transcription inhibition(Hg)

~ DNA -. mRNA -. Protein synthes is

t t DNA damage Translation inhibition (Hg. Pb, Cd & As) (Hg, Pb & Cd)

Fig. I - Heavy metal toxicity mec hani sms to microbes

- 1

RAJ ENDRAN el at.: MICROB ES IN HEAVY METAL REMEDIATION 939

Table 4-Mechanism of metal to lerance in microorganisms

Metal

AsO; AsO/ & SbJ+ Cd2+-& Zn2+ Hg2+ Co2+ & Ni2+ Cr0 42. Cd2+ Cd2+ & Zn2+ cr02. Cu2+

Tolerance mechanism

Anion effl ux (ATPase) Efflux (ATP ase) Reduction Efflux Decreased uptake Cat io n effl ux Decreased uptake DNA damage.

regulatory transcriptional factor-l (MTF-l ) was essenti al for basal and heavy metal induced transcription of the stress- responsive metallothionein-I and metallothionein-II. Metallothionein like proteins have been isolated from the Cyanobacterium, Syneococcus spp as well as Escherichia coli and Pseudomonas putida4 ]. Plasmid-encoded energy dependent metal effl ux systems in volving ATPases and chemiosmotic ion/ proton pumps are associated to Ar, Cr and Cd resisstance in Staphylococcus aureus, Bacillus sublilis, Listeria spp, E.coli, A.eutrophus, P.pulida, some Cyanobacteria, fungi and algae 7 . E.coli use a two-component membrane bound ATPase complex, ArsA and ArsB responsible for the active efflux of arsenite form the cell. In contrast Gram-positive bacteri a lack the ArsA protei n44.

Genetics of metal resistance Recent environment pollution with anthropogenic

sources of metals has increased the need for research concerning microbial metal res istance as well as remedi ati on. Large plasm ids (165-250 kb), encoded specific metal conferring res istance to a variety of metals including Ag+, As02', AsO/ , Cd

2+, C02+, CrO/, Cu2+, Hg2+, Ni2+, Pb2+, Sb3+, Te02.] , TI+, and Zn2+ 45. Metal resistance systems have been encoded by chromosomal genes in so me organisms like, Bacillus.spp (Hg- res istance) and E.coli. (A r-efflu x). Roane and Pepper7 categori zed mechani sm of metal resistance as I) general and do not requ ire metal stress 2) dependent on a spec ific metal for ac ti vation and 3) general and are acti vated by metal stress.

The pl asm id-borne cadA gene encodes a cadmium specific ATPase in severa l bac terial genera, including Staphylococcus, Pseudomonas, Bacillus and E. coli. Schmid and Schegel46 reported the czc and nee operons are responsible for cadmium resistance in Alcaligenes eutrophus CH34 (now renamed as Ralstonia eUlropha) and in A. xylosoxidans, respectively. The genetic determinants for heavy metal resistance in A.eutrophus CH34 are present in

two plasmids, pMOL 28 and pMOL 30. Nickel resistance is encoded by pMOL 28 (163 kb) which occur by an efflux pathway via cation proton-anti porter chemiosmotic system47 . This removes the toxic metal , which is accumulated by the uptake of essential divalent cations (e.g. Mg2+, Mn2+) . Taghavi et al.48 showed that nickel resistance is inducible and is due to an energy-dependent efflux system dri ven by chemiosmotic proton-anti porter system.

Genetic engineering for metal."emediation Genetic engi neering allows introducti on of desired

traits into cell s to metal clean up techniques, and th is approach has already been used to construct cells for the bioremedi at ion of mercur/ 9. Kri shnasamy and Wilson5o constructed an E.coli strai n th at accumulated Ni 2+ by introducing the ni xA gene (cod ing for a nickel transport system) fro m Helicobacter pylori into E. coli 1M 109 that expressed a glutathi one S-trans-ferase pea metallothioneine fusion protein . The recombinant E.coli strain acc umulated four times more ni ckel than the wild type. Bang et at. 51

engineered E.coli to produce sulphide by heterologous expression of the thiosulphate reductase gene from Salmonella entenltca, which enhanced protein synthesis leading to the precipitation of cadmium sulphide. Metal resitant R. eUlropha isolate, engineered to produce metall othionein accumulated more Cd2+ than its wild type counter part , and offered tobacco plants some protec tion form Cd2+ when inocul ated into contaminated soil 52. Studies of Pazirandeh28 showed periplasmic ex pression of a Neurospora crassa metallothonein in E. coli generated cell s that were superior to bacteria with cytoplasmic metallothionein locati on in terms of metal ion adsorption. Kotriba el al. 53 engineered metal binding peptides that contain either histidines (GHHPHG)2 (HP) or cystines (GCGCPCGCG) (CP) into LamB and expressed on the surface of E.coli. Surface display of CP and HP increased the bioaccumulation fo ur and two fo ld, respectively.

54 d b' Samuelson el al. reporte recom 1nant Staphylococcus xylosus and S. carnosus, had gained Ni2+ and Cd2+ binding capacity and suggested that they could be used in bioremediation of heavy metal s. They evaluated surface display systems for expression of two different polyhistidyl peptides, Hi sr Glu-His3 and Hi s6, which had good metal binding acti vity. The nickel resistant bacteria from anthropogen ically polluted biotypes and naturally nickel-ri ch soil s have been hybridized with various probes carrying cnr

940 INDI AN J EXP BIOL, SEPTEMBER 2003

from pl asmid pMOL28 (A. eutrophus C H34), nee from pTOM8 (A. xylosoxidans 3 1 A) nre fro m pTOM8 (A. xylosoxidans 3 1A) and nre fro m Klebsiella oxytoea 15788. With respect to the ir hybridi zati on signals, the nicke l res istant determinants of the stra ins could be assigned to enrlnee type, enrlncc-nre type Klebsiella oxytoca type and o thers showing no hybridizatio n46 . Bacteri a l communities in soil amended fo r many years w ith sewage sludge that conta ined heavy metals were assessed using molecul ar tools such as , rRNA analys is, F ISH (Fluoresence in-situ hybridi zatio n), cloning and sequencing55 .

Biosorption Biosorption, uti li zati on of inex pensive dead or li ve

mi crobi al biomass to sequeste r metals from industria l effl uents has ga ined impo rtance in recent years due to their good performa nce, low cost, specific ity, minimu m s ludge generation and amenabili ty fo r repeated use34.56. Severa l ac ti ve groups of ce ll

MICROBE

Passive mechanism

Ex tra ce ll ular complexation (CilrobaCler spp)

Extra cellular polymeric substance (S.aure lls)

Polysaccharides (Z rallligera) Proteins (A. bisporous) Nuc leic acid

constituents like acedamido group of chitin , structural polysaccharide of fung i, amine (amino and peptidoglycosides), su lfhydra l and carboxy l groups in protein , phosphodiester (te icho ic ac id), phosphate, hydroxy l in polysaccharides, parti c ipate in biosorptio n57 . The genera l microbi al mediated heavy meta l sorption mechani sms a re in the Fig. 2. Hernandez et ai. 2 iso lated three species of bacteri a belonging to fa mil y Enterobacteriaceae, whi ch were capable of accu mulating nickel and vanad iu m. Biosorption fo r metal re moval has been stud ied ex tensi ve ly using various species of li ve and inacti vated biomass of bacteri a, algae, fung i or yeast 1.57. T he techno logy in vo lving surface complexatio n, ion exchange and micropreceipitati on is a potenti al a lte rnati ve to current metal treating processes that ex tend the durati on of exposure of the clean up crews58 . Research in the area of heavy metal removal fro m wastewater and sed iments has focused on the development of microbi a l materi als w ith increased affinity, capac ity and selecti vity for target

Rhamnolipids (P.ae ruginosa)

Intrace ll ular accumu lation (Pseudomollas spp)

Metal lothionein (Synechococcu.I' spp)

Precipita tion (CilrobaCler spp)

Effl ux pump (A.e lllrophus)

Active mecha nism

Ion exchange reaction (S.pia lellSis)

Complexation with oxygen and nitrogen ligands (P. pUl ida)

Destruction of complexing ligands (AcinclOiJacler spp)

Volatili za tion (Fu ngal spp)

Fi g. 2 - Mechanism of microbial metal to lerance

RAJENDRAN el al.: MICROB ES IN HEA VY METAL REMEDIA TlON 94 1

metals28,54 . Fungal bio mass is finding increas ing appli cation in current biotechnological procedures for the cleanup of nickel-polluted effluents and fo r the recovery of metal ions I. Rajendran et al. 59 reported A.niger mutant M 3 capable of absorbing 50% more nickel at 1.7 mM concentration than its parent strain . The waste fungal biomass from industrial fermenters may prove to be cost effecti ve meta l biosorpti ve agents on a large scale6o. Fouvert and R O UX61 studied the use of myce li a o f Mucor nicheri, Aspergillus niger and Pencillium chrysogenum fro m fermente rs in the removal of nickel, z inc, cadmium and lead by bio-adsorption. Gadd31 reported chemical modification of biomass mi ght create deri vati ves with a ltered meta l binding abilities and affiniti es. Pseudomonads have been shown to be effic ient in heavy metal bioaccumulation fro m polluted effl uents in the immobilized state62.63 .

Immobilization for metal remediation Biosorpti on technique for metal removal and

accumulation can be even w ith a low degree of understanding of the metal binding mechani sms. However, a better understanding of the technique will be useful fo r effect ive and optimi zed application of the method64 . Free ce lls can provide valuable in fo rmation in laboratory experimentatio n but have li mi tations in industria l applications65 . The use of free ly suspended mi crobi a l bio mass suffe rs with d isadvantages li ke small partic le s ize, low mechanical strength and di fficulty in seques tering biomass and effluent. The immobilized bioadsorbent bi omass packed in sorption columns, which are effecti ve fo r continuous remova l of heavy metals can operate o n cycles consisting of loading, regeneration and rinsing66 . Ibanez and U metsu67 have demonstrated the ability of protonated alg inate beads in the removal of chromium, copper, zinc, nickel and cobalt ions from dilute aqueous solutions. Bio mass immobili zed in a range of inert materi als like, s il ica, polyacry lamide, po lymethane and polysul fo ne has been used in a vari ety of bioreactor configurati ons, inc luding rotating bi ological contractors, f ixed reactors, tri ckle filters, flu idi zed beds and a ir li ft bio reacto rs65 . Karna et aL. 68 immobilized Phormidium valde rianum BD U 3050 ] in polyv iny l foa m and used for the removal of C ?+ C 2+ C ?+ d N '?+ 63 a- , 0 , u- an 1- . Ashtana et al. reported that Ca-alginate immob ili z ing agent is very effecti ve in nickel biosorpti on and used ino rgani c sa lts like NaCI and Ca (N03)2 fo r desorpti on of nickel fro m immobili zed microbia l bi o mass. HC I and EDTA are

the most effic ient desorption agents for nickel removaI62,68.

Plant-microbe interaction in metal remediation Heavy metal tox ic ity to plants can be reduced by

the use of pl ant growth promoting bacteri a, free-li ving soil bacteria that exert some benefic ial effects on plant development when they are applied to seeds or incorporated to seeds70 . Plants can acce lerate bioremediation in surface so ils by the ir abil ity to stimulate so il microorganisms through the re lease of nutri ents fro m the soil and transpo rt of oxygen to the rhi zosphere7o. Plant growth pro moting bacteri a that contain ACC deaminase may act to insure that the ethy lene level does not impair root growth and facilitate the formation of larger roots, which enhance seedling surviva l7 1. Burd et al. 72 reported that a metal-res istant soil bacterium Kluyvera ascorbata S UD 165 pro moted the growth of cano l a (Brassica campestris) in the presence of high concentratio ns of nickel and the increase in growth rate is due to the abi lity of the bacterium to lower the level of ethy lene stress in the seedlings. They furth er iso lated siderophore over producing mutants, K. ascorbata 165/26 which was able to prov ide suffic ient ions for the growth of Indian mustard (Brassica j uncea) and tomato (Lycopersicon esculentum) in the presence of high concentrations of Ni, Pb, and Zn . Studies of Bu rd et aL. 73 suggested the best method to prevent plants fro m becoming chloroti c in the presence of high levels of heavy metals was to provide them with a siderophore-producing bacterium, Kluyvera ascorbata SUD 165. Mycorrhi zal fung i a lso reduce metal tox ic ity to the ir host pl ant by bind!ng heavy meta ls to their ce ll wall o r surrounding po lysaccharides. Binding sites w ithin cell s of ectomycorrhi zal fung i are metallothi onein-like prote ins, which have high cys tine content and perhaps polyphosphate granules7o.

Field applications The mos t important bio techno log ical application of

meta l-microbe interactio n is in bio leaching, bioremediatio n of po lluted sites and minera li zati on of po lluting orgninc matter l6 l) ranium is extracted fro m uranite ore th rough bi oleaching by indirect action with T.Jerroxidans . In thi s case, ferri c iro n in acidic so lution, which the organi sm generate when ox id izing pyrites (FeS2), ox idi zes in so luble UO to soluble UO/+74 . T he ex traction of meta ls such as Co, Mo, Ni, Pb, and Z n fro m sul f id ie o res by bi oleaching is technbically feas ible. Gencor (South Africa) is cun'ently

942 INDIAN J EXP BIOL, SEPTEMBER 2003

developing tlie BioNIC process to recover nickel from low-grade sulfide ores75 . Signet Technology Inc. and Signet engineering Pvt. Ltd . (Western Australia) are cUlTently developing a process to recover cobalt from pyrite ore from the kares cobalt project in Uganda76. Various microbially reducible metals, especially ferric iron in complexed form to keep it soluble at circum neutral pH, can be used as terminal electron acceptors in in s itu anaerobic bioremedi ation of sites polluted with tox ic organics77 . It is possible to bioremediate in situ sites polluted with chromate or dichromate [Cr (Vl)] by stimul ating the reducti on of the Cr (V I) to Cr (Ill) by bacteri a25 . Fungi can convert oxidized selenium to volatite-methylated selenides for escape into the atmosphere, a removal by volatilizat ion 78 Beseker79 reported Penicillium fu.niculasul1l was ab le to extract more than 50% Ni and 75% Zn from test solu tions containing 100 mg rt of metal at pH 6.6 and 6.5 respectively. A potent algal biosorbent Alga SorbT.Y1 developed using a fresh water alga ChIarella vu.lgaris and AMT-BIOCLAIMTM (MRA) developed using Bacillus biomass are used to treat wastewater and metal recovery respecti vel/ 6 .

Future prospects Microbes can significantly affect the di stribution of

metals in the environment , since they have developed means to use them for their benefit. This clearl y holds promise for effecti ve, economica l and eco-friendl y metal bioremediation technol ogy for industri al ex ploitati on and pollution free environment. A good and efficient metal biosorbent can repl ace the commercial ion exchange res ins that have been used convent ionally for metal removal. However, the bas ic knowledge of microbi al metal bioremediation mechani sm is inev itable for the development of commercially viable potent biosorbent. Although several mi crobi al metal bioremedi at ion approaches are establi shed to combat heavy metal pollution from anthropogenic and natural sources, none is yet in wide spread use. Innovative, economicall y feasible and novel biomass regenerati on and conversion of the recovered metal into usable form are the best options to attract more usage of biosorbents. It is time to II1ttlate more comprehensive interd isciplinary approach between biotechnologists and metallurgists to bring lab sca le bioremedi ation process to land scale technology that will be acceptabl e for industriali sts.

Acknowledgement One of the authors CPR) is indebted to UGC, New

Delhi fo r the award of Teacher fe ll owship for doctoral

studies and to the Management of Vivekananda College, Thiruvedagam, Madurai for deputation under F.I.P. program.

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