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 app lication for metal
accumulation/detoxification. This article summari zes the potentials of microbes in metal remedi ation .

Keywords: Bioremediation techniques, Heavy metal contamin ation, 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 wastes 3, automobile
4 ID
ernissions , mining activit/, and agricultural
6
practices . 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 metal s into three
classes on the basis of their biological function s 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 metal s (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 metals 8 .
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: 0452-545 8478; 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
e nzyme catalysis, nutrient transport, protein structure,
charge neutraliziation and control of osmatic
pressure9. Olson et al. reported incorporation of
nickel in four microbial enzymes involved in
ureolysis, hydrogen metabolism, methane biogenesis
and acetogenesis. A number of heavy metal s 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 th at 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 metal s 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

(Cu +, Cd 2+, Pb 2+ & Ca2+) and in reduced or a naerobi c

2
their pathological effects, whi ch pose health hazard to
envi ronment (T abl e I ) 14. conditi o ns (0 to -400 mv) they are fo und as CuS ,
7
PbS , and CdC03, precipitates . At low soil pH, the
Metal availability me ta l bioavailability increases due to its free ioni c
5 spec ies and in contrast, hig h so il pH dec reases due to
Sposito' categori sed me tal s as bioavail abl e
(prec ipitated, non- sorbed and mobil e) and non- in so luble metal minera l phosph ate a nd carbo nate
bioavailable (precipitated , sorbed , and non-mobile) form ation . Ho we ver, in contrast to other metal
metals. In nature , metals and Il!etalloids ex ist mostl y bioavailabilities, at hi g h pH range o f 5-9, ni c ke l may
as catio ns, oxy anion s, or both in aqueoll s soluti o n and be adsorbed o n iron a nd manganese ox ides, o r fo rm
mostl y as salts or oxides in crystalline (minera l) form compl exes with inorganic li gands (OH -, S042- , cr o r
7
o r as amorph ous prec ipitates in solubl e form . ' 6 . The NH3)21.
mobility of metals as hydrated io nic salts is depe ndent
on two factors 1. The meta llic e le ment that is Metal microbe interaction
parti c ipating as positi vely charged io ns (cati o ns) and Microbi al interacti o ns w ith heavy meta ls were
2. The one, which makes up negati vely charged rev iewed by, Ehrli ch ' 6 and Beveridge a nd Doy le 22 .
compo nent o f the salt. The cati o nic/a ni o nic so lubility Mi crobi al interacti o ns with sma ll qu antiti es of meta ls
re lati ons hip is in T abl e 2 . Geoche mi cal fo rms o f o r metalloids do not exert a maj or impact o n metal o r
heavy metals in soil affect th e ir so lubility , which metalloid d istributi o n in the e nviro nme nt, whereas
direct ly influence the ir bi oavail abili ty ' 7. Le na and interactions with larger quantiti es are required in
l8
Rao repo rted mo bility and bioava il ability of metals energy metabo li sm, fo r in stance, to ha ve no ti ceabl e
in so il s is in the orde r of Zn > Cu > Cd > Ni , based on impac t ' 6 . Howe ver, due to stron g io ni c nature metals
their so lubility and geoche mi ca l fo rms. The fa te of
tox ic meta ls in so il s depe nds ma inl y o n the initi al Table 2 - Summary of cati on/a ni on solu bi li lY relati onshi p of
metals
chem ical form o f the metal even thou g h the
enviro nmental and edaphi c conditi o ns s uch as pH , Anions Catio ns Solu bil ity
redo x statu s, and so il o rgani c matte r conte nt have All ani ons Na+, K+, NH/ Solub le
sig ni ficant influence ' '! . Studi es of Sa uerbeck and NO ;- , NO -2 ' C 2H30 2, All metals Solub le
Ri etz 20 have indicated the ex iste nce of different
Mn0 4, CIO ~ , CIO ~
bi nding fo rms and strong ly p H depenent so lubility
CI- , Br- , 1- All metals except Solu ble
effec ts of the trace e le ments. Soil cation exchange Pb 2+ , Ag+, Hg2+
capacity (CEC) affect meta l bi oavail ibilty and it SO ~ - All metals except Solub le
depends o n orga ni c matter and cl ay co nte nt o f the Ba 2+, Sr2+, Pb2+
so il. T he tox ic ity of metals within so ils with hi g h 0 2-, S2-, OW All metals except Insolu ble
CEC is low even at hi gh to tal me ta l co ncentrati o n and Ca 2+, Ba 2+, Sr2+
7
in contrast the toxic ity is vice-versa in lo w CEC . CO ~ - , PO ~- , SO ~- All metalli c salts except Inso lub le
Under ox idi zed o r aero bi c co nd iti o n (+ 800 to mY ) a BO j- , F , SiO j-
Na+, K+, NH/
"metals are usua ll y fo und as so lubl e cati o ni c fo rm s

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

Metal Use Effect

Hg Coal. vinyl chl orides. electri ca l balleri es

Pb Pl as ti c, paint, pipe, balleries, gasoline, auto ex haust Neurotoxic
A I' Pesti cides, coal. deterge nts Live rcirrohosis, meni al disturbance, cancer, ulcer and hypokerot os is
Cd Ferti I ize r, pl astic, pigment Kidn ey damage, inju ry in cns and mental retardati on
Cr Tannin g, paints, pigmenl , fun gicide Nephritic, cancer and ulcerati on
Zn Fertil izer Vom iting, renal damage and cra mps
Co Vilam in B- J2 Diarrh oea. low blood press ure and paralys is
Se Coa l, sulfur Damage of li ver, kidney , spl een and nervo usness
Ni Electro plating Teratoge ni c, carcinogenic, genotox ic and mu tage n
Bc Coal, roc ket fuel Carcinogen, ac ute and chron ic poison
 RAJENDRAN e( al.: MICROBES IN HEAVY METAL REMEDIATI ON
bind to many cellul ar li gands and di spl ace nati ve
essential metals fro m the ir norma l bindin g sites,
7 28
which are tox ic . E ubacteri a and archaea are abl e to
ox idi ze Mn (II), Fe (II), C o (Ill), AsO ; , Sea o r reduce
Mn (IV), Fe (Ill) C o (II) AsO ~ - , SeO ~ o n a large
scale and conserve energy in these reac ti o ns '7. So me
microbes reduce io ns such as Hg2+ o r Ag+ to HgO and
AgO respec ti vely, but do no t conserve energy fro m
these reacti on 23 . Prokaryotes methy late meta l and
meta ll oid compo unds produc in g co rrespo ndin g
24
volatil e metal deri vati ves . The bacteri a l ox id ati o n o f
AsO ; to AsO !- by a stra in o f Alcaligenes faeca lis
and reducti o n of CrO ;- to Cr (OH h by Pseudomonas
j luorescens LB 300 or Enterobacter docae are examples
of redox reacti o ns in vo lving e nzy mati c mi crobi al
detoxifi cati on of harmful meta ls o r metall o ids 25 .
Microbes secrete in organi c metabo li c produ cts such
as sulfide, carbo nate o r ph osph ate ions in the ir
respiratory metabo li sm and with them prec ipitate tox ic
26
metal ions as a form of no n-enzy mati c detoxificati on .
In nature mi crobes immo bili ze me tals throu gh ce llul ar
sequestrati on and acc umul ati on, or through extracelliul ar
.. . '7
prec lpltatJOn - .
Conventional method of heavy metal removal from
soil and water
Vari o us methods are avail abl e fo r the re mova l and
manage ment of heavy meta ls, w hi ch in vo lve techni ca l
inputs. C urrent meth ods used fo r treating so il s
contamin ated with tox ic meta l are 1) Land fillin g: the
excavati o n, transpo rt and de pos iti o n o f co ntamin ated
soil in a permitted hazardo us was te land , 2) Fi xati o n:
the che mi ca l process ing of soil to immo bili ze the
metals, usuall y fo ll o wed by treatment o f the so il
surface to e limin ate pe netrati o n by wate r and 3)
Leaching: using ac id so luti o ns as pro pri etary leaching
agent to desorb a nd leach meta ls fro m so il fo ll owed
by the return of cl ean so il res idue to site '9 .
Approaches to reduce heavy metal co nta min ati on
in water include: 1) prec ipitati o n or fl occ ul ati o n,
foll owed by sedime ntati o n and di sposal of the
resulting sludge, 2) io n exc hange, 3) reverse os mos is,
4) microfiltrati o n, 5) e lectrodi alys is a nd 6)
evapo ration . Apart fro m these, o ther methods like
foa m flo atati o n, liquid me mbrane technique, so lve nt
extractio n and crysta lli zati o n ca n a lso be e mpl oyed .
Bioremediation techniques
The goal of mi crobi al re medi ati o n of heavy meta l
contaminated so il s and sedime nts are to immo bili ze
937
the metal in situ to reduce metal bioava il ability and
mobility or to re move the metal from the so il.
Pazirandeh et al. desc ribed the metaboli c path ways
res ulting in bioprecepitati o n o f heavy metals o r the ir
bi o tran sform ati o n. Lo w cost and hi g her e ffic iency at
lo w meta l co ncentrati o ns make bi otechno log ica l
process very attracti ve in compari so n to ph ysico-
29
che mi cal methods fo r heavy meta l re moval . M etal
re medi ati o n strateg ies using mi croo rgani sms can
minimi ze the bi oava il ability and bi o tox ic ity of heavy
3o 31
metals . . Biostimul atio n, stimul atio n of viable nati ve
mi crobi al po pul ati o n, bi oaug ume ntati on, artif ic ial
introdu cti o n o f viabl e po pul ati o n, bi oacc umul ati o n
use o f live cell s, and bi osorpti o n, use of dead
mi c robi al bi o mass, are the cost-effec ti ve inn ova ti ve
bi o re medi ati o n techno log ies. Bio log ica l approac h fo r
meta l detox ifi cati o n affo rds the po tenti al fo r se lective
re moval of tox ic meta ls and o peratio n fl ex ibility and
easy adaptability fo r in situ and ex situ appli cati on in
3o
a range o f bi o reacto r confi g urati o ns . In the past few
decades, new meta l treatment and recovery techniq ues
based o n bi osorpti o n have been e xplored usin g both
dead and living mi c robi al bi o mass with re marka ble
e ffi c ie ncy. Prokaryotic and euk aryoti c mi crobes are
capabl e of acc umul ating metals by bindin g th em as
22
cati o ns to the ce ll s urface in a pass ive process .
Microbes for metal remediation
Th e mechani sms by w hi ch metal io ns bind to the
ce ll surface include e lectrostati c interac ti o ns, Va n der
Waals fo rces, covalent bo nding, redo x interacti ons,
and ex tracellul ar prec ipitati o n, o r combin ati o n o f
these processes 32 .The negative ly charged g ro ups
(carbox yl, hydrox y l, and ph ospho ryl) o f the bacteri al
cell wa ll adsorb meta l catio ns, w hich are then retained
33
by min eral nucleatio n . Bi osorpti o n studi es o f U, Z n,
Pb, Cd, Ni , Cu, Hg , Th , Zn , Cs, A u, Ag, Sn and M n,
sho wed th at th e ex te nt o f so rpti o n vari es markedly
with the metal as we ll as with the mi croo rgani sms
(T abl e 3).
Surfactants s uch as rh amnolipid produced by
P. aerugin osa sho w spec ifi c ity for certain meta ls uch
as Cd and Pb. Hi ghe r mo lec ul ar we ig ht (- 10 6)
bi oemul sifi ers such e mul san, can also aid in metal
35
remov al . Studies of Sand et a f. 36 revea led th at
ThiobacilUus f erroxidans and Leptospirillum
fe rroxidans are capabl e of ox idi z in g iro n and s ulfur.
Joerger et al. 37 repo rted that metal acc umul ating
bacterium Pseudomonas stutzeri AG 259 is capab le of
produc ing sil ver based sin gle crystals, whi ch can
reduce the to xi c ity of meta ls.
938 INDI AN J EXP BIO l , SEPTE MB ER 2003

Mechanisms of metal tolerance spec ific actI vIty. Mi croorgani sms can affec t heavy
O rgani sms respo nd to heavy metal stress using metal concentrati o ns in the e nvironment bec au se they
di ffe rent defense systems (Fig. 1) such as exc lus io n, ex hibit a strong ability fo r metal removal from
compartmenta li zati on, fo rmati o n of co mplexes and so lution ; thi s can be achieved th roug h e ither
sy nthesis of binding prote in s like metallo thione ins enzy matic o r non-enzymatic mechanj sms39 . A voidance,
38
(MTs) and phytoche latin s (PCs). Ochari has di vided restricti o n of meta l entry into the ce ll, e ither by
general tox ic ity mechani sm fo r meta l io ns into three reduced uptake/acti ve efflu x o r by th e fo rmati o n of
categories, 1. blocking the essenti a l bi olog ical co mplexes o utside the ce ll a nd sequestrati o n,
funct io nal groups o f bi o mo lecul es espec ia ll y pro te in s reducti o n of free io ns in the cy toso l e ither by
and e nzy mes, 2. di spl ac ing th e essenti al meta l ion in sy nthes is of li gands to achieve intrace llu lar che lati o n
bi o mo lec ul es and 3. modifyin g the acti ve or by co mpatme nta li zati o n are th e two major
conformati o n of bi omo lec ul es res ul tin g th e loss of strateg ies o f o rgani sms to pro tect themselves again st
heavy metal tox ic it/o. The general mec hani sms of
Table 3 - Exam ple of microorgani sms th at take up heavy metals meta l tolerance in mi cro bes are shown in Table 4.
Microorgan isms Elemen ts Uptake
(% dry wt) Metallothioneine
Meta ll othi o ne ines, di scovered abo ut 45 years ago,
Zooglea spp. Co 25
Ni 13 pl aya centra l ro le in heavy meta l me tabo li sm and in
Citrobacte r spp Cd 170 the management of vari ous forms of stress.
U 900 Meta llo thi o nein es (MTs) are lo w molec ul ar weight
Ba cillus spp Cu 15 (6-7 kDa), cystine- ri c h prote in s, di vided into three
Zn 14
Chlorella vulga ris Au 10 di ffe re nt cl asses o n the bas is of the ir cystine content
Rhizopus arrh iZ.lIs P 10 and s tru~ture . The C ys-Cys, Cys-X-Cys and C ys-X-
Ag 54 X-Cys, mo tifs (in whi ch X de notes any amino acid )
Hg 58 41
are characteristi c a nd inv ari ant fo r metall o thi o ne ine .
Aspergillus niger Th 19 42
Studi es of Li chtl e n and Schaffner show meta l

Protein denaturation Inhibition of cell division

(Hg, Pb & Cd ). (Pb. Cd. Hg & Ni)

/
MIC ROB ES

Cell membra ne disr uption Inhibiti on of enzyme acti vity ]

(Hg, Pb. Zn. Ni. C u &Cd) (Hg. Pb. Zn, Ni. Cu & Cd)

Transcription inhibition(Hg)
~
DNA - . mRN A - . Protein synt hes is

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

Fig. I - Heavy metal toxic ity mec hani sms to microbes

 RAJ END RAN el at.: MICROB ES IN HEAVY METAL REMEDIATION
Table 4-Mechanism of me tal to lerance in microorganisms
Metal Tolera nce mechanism
AsO; AsO/ & Sb J + Anion effl ux (ATPase)
Cd 2+-& Z n2+ Efflux (AT P ase)
Hg2+ Reducti on
Co 2+ & Ni2+ Efflux
Cr0 42. Decreased uptake
Cd2+ Cd2+ & Zn2+ Cat io n effl ux
cr02 . Decreased uptake
Cu 2+ DNA damage.
regul atory transcriptional factor-l (MTF-l ) was
essenti al for basal and heavy metal induced
transc ripti on of the stress- responsive metallothionein-
I and metallothi onein-II. Metallothionein like proteins
have been isolated from th e Cyanobacterium,
Syneococcus sp p as well as Escherichia coli and
4
Pseudomonas putida ]. Pl asmid-encoded energy
dependent metal effl ux systems in vo lving ATPases
and chemiosmotic ion/ proton pumps are assoc iated to
Ar, Cr and Cd resisstance in Staphylococcus aureus,
Bacillus sublilis, Listeria spp, E.coli, A.eutrophus,
7
P.pulida, so me Cyanobacteria, fungi and algae .
E.coli use a two-component membrane bound ATPase
co mpl ex, ArsA and ArsB respo nsible for th e active
efflux of arsenite form th e cell. In co ntrast 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 plasmids (165-250 kb), encoded
specific metal co nferring res istance to a vari ety of
metals including Ag+, As0 2', AsO/ , Cd 2+, C02 +,
- 1 CrO/, Cu 2+, Hg2+, Ni 2+, Pb 2+, Sb 3+, Te0 2 .] , TI+, and
Zn 2+ 45. Metal resistance systems have been encoded
by chromosomal genes in so me orga nisms like,
Bacillus.s pp (Hg- res istance) and E.coli. (A r-efflu x).
Roane and Pepper7 categori zed mec hani 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-bo rn e cadA gene encodes a cadmium
specific ATPase in severa l bac terial genera, including
Staphylococcus, Pseudomonas, Bacillus and E. coli.
Schmid and Schegel 46 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 geneti c determin ants for heavy
metal resistance in A.eutrophus CH34 are present in
939
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+, Mn 2+) . Taghav i
48
et al. showed that nickel resistance is inducible and
is due to an energy-dependent effl ux system dri ven by
chemiosmoti c proton-anti porter system .
Genetic engineering for metal."emediation
Genetic engi neerin g 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 bi oremedi at ion of mercur/ 9 . Kri shnasa my and
Wilso n5o constructed an E.coli strai n th at acc umul ated
Ni 2+ by introd ucing the ni xA gene (cod ing for a
ni ckel transport syste m) fro m Helicobacter pylori into
E. coli 1M 109 that ex pressed a glutathi one S-trans-
ferase pea metallothioneine fusion protein . The
recombinant E.coli strain acc umul ated four times
more ni ckel th an the wild type. Bang et at. 51
engineered E.coli to produce sulphide by heterologous
expression of the thiosulph ate reductase gene from
Salmonella entenltca, which enhanced protein
sy nthesis leadin g to the precipitati on of cadmium
sulphide. Metal resitant R. eUlropha isolate,
engineered to produce metall othi onein accumul ated
more Cd 2+ th an its wild type counter part, and offered
tobacco plants so me protec ti on form Cd 2+ when
inocul ated into contamin ated soil 52. Studies of
Paz irandeh28 showed peripl asmic ex pression of a
Neurospora crassa metall othonein in E. coli generated
cell s th at were superior to bacteria with cytoplasm ic
metallothi onein locati on in term s of metal ion
adsorption. Kotriba el al. 53 engineered metal binding
peptides that co ntain either histidines (G HHPHG )2
(HP) or cystines (GCGCPCGCG) (C P) into LamB
and expressed on th e surface of E.coli. Surface
display of CP and HP increased the bioaccumulation
fo ur and two fo ld, respectively.
Samuelson el al. 54 reporte d reco mb'1nant
Staphylococcus xylosus and S. ca rnosus, had gained
Ni 2+ and Cd 2+ binding capacity and suggested that
they co uld be used in bioremediation of heavy metal s.
They evaluated surface di splay systems for ex pression
of two different polyhistidyl peptides, Hi sr Glu-His 3
and Hi s6 , which had good metal binding acti vity. The
ni ckel resistant bacteria from anthropogen icall y
polluted biotypes and naturall y ni ckel-ri ch soil s have
been hybridi zed with various probes carrying cnr
940 INDI AN J EXP BI OL, SEPTEMBE R 2003

fro m pl as mid pMOL28 (A. eutrophus C H34), nee
from pTOM8 (A. xylosoxidans 3 1A) nre fro m
pTOM8 (A. xylosoxidans 3 1A) and nre fro m
Klebsiella oxytoea 15788. With res pect to the ir
hybridi zati o n sig na ls, the ni cke l res ista nt determina nts
of the stra in s coul d be assig ned to enrlnee ty pe,
enrlncc-nre type Klebsiella oxytoca type and o thers
46
show in g no hybridi zatio n . B acteri a l co mmuniti es in
soil amended fo r many years w ith sewage sludge that
conta ined heavy metals were assessed usin g
mo lecul ar too ls such as , rRNA ana lys is, F ISH
(Fluoresence in -situ hy bridi zati o n), clonin g and
seq uenc ing 55 .
Biosorption
B iosorpti o n, utili zati o n of in ex pensive dead or li ve
mi crobi al bio mass to seq ueste r metals fro m indu stria l
effl ue nts has ga ined impo rtance in rece nt years due to
their good performa nce, low cost, spec ifi c ity,
minimu m s ludge gene rati o n and a menabili ty fo r
34
repeated use .56 . Severa l ac ti ve groups of ce ll
constitue nts like aceda mido gro up of c hitin , struct ural
po lysaccharide of fun g i, amine (a mino and
peptidog lycosides), su lfhydra l and carboxy l groups in
protein , phosphodi ester (te icho ic ac id), ph os ph ate,
hydroxy l in po lysaccharides, parti c ipate in
57
biosorpti o n . T he genera l mi crobi al med iated heavy
meta l so rpti o n mecha ni sms a re in th e Fig. 2.
Hern andez et ai. 2 iso lated three spec ies of bacteri a
be lo ngin g to fa mil y Entero bacteriaceae, w hi ch were
capabl e of accu mul ating ni cke l and va nad iu m.
Bi osorpti o n fo r metal re mova l has been stu d ied
ex te nsi ve ly usin g vari o us s pec ies of li ve and
inacti vated bi o mass of bacteri a, algae, fung i o r
yeast 1.57. T he techno logy in vo lving s urface
compl exati o n, io n excha nge and microprece ipitati on
is a pote nti al a lte rn ati ve to c urrent metal treating
processes that ex tend the d urati o n of expos ure of the
clean up crews 58 . Researc h in th e area of heavy metal
re moval fro m wastewater a nd sed ime nts has foc used
o n the development of mi crobi a l materi als w ith
increased affinity, capac ity and selecti vity for target

Rhamno lipids
(P.ae ruginosa)

Intrace ll ular accumu lation Metal lothi onein

(Pseudomollas spp) (Synechococcu.I' spp )
MICROBE
Precipita tio n
(CilrobaCler spp)

Vo latili za tion
Effl ux pump
(Fu nga l sp p)
(A.e lllrophu s)
Passive mec hanism

Ac ti ve mecha nism

Ex tra ce ll ular complexati o n

(CilrobaCl er spp)

Ion exchange reactio n

Extra cellular polymeric substance (S.pia l ellSis)
(S.a ure ll s)

Complexa tio n with oxygen and

nitrogen ligands (P. pUl ida)

Polysacc harides (Z rallligera)

Proteins (A. bisporous) Destruction of co mp lexing
Nuc leic acid liga nds (AcinclOiJacler spp)

Fi g. 2 - Mec hanism of mi crob ial metal to lerance

 RAJENDRAN el al.: MICROB ES IN HEAVY METAL REMEDIATlON
metals 28 ,54 . Fungal bi o mass is finding increas ing
appli cati on in current bi otechno log ical procedures for
the cleanup of ni ckel-po lluted efflue nts and fo r the
recovery of metal io ns I. R aj e ndran et al. 59 repo rted
A.niger mutant M 3 capabl e of absorbin g 50% mo re
nickel at 1.7 mM conce ntration than its pare nt strain .
The waste fungal bi o mass from industrial fermenters
may pro ve to be cost effecti ve meta l bi osorpti ve
61
agents on a large scale 6o . Fouvert and R O UX studi ed
the use of myce li a o f Mucor nicheri, Aspergillus nige r
and Pencillium chrysogenum fro m ferme nte rs in the
removal of nickel, z inc, cadmium and lead by bi o-
31
adsorpti on. G add repo rted chemi cal modificati o n of
bi o mass mi ght create deri vati ves with a ltered meta l
binding abilities and affiniti es. Pseudo mo nads have
been show n to be effic ie nt in heavy metal
bi oaccumul ati o n fro m po lluted effl uents in th e
immobili zed state 62 .63 .
Immobilization for metal remediation
Bi osorpti on tec hnique for metal re moval and
accumul ati o n can be e ve n w ith a lo w degree of
understanding of the metal binding mechani sms.
However, a better understandin g of the technique will
be useful fo r e ffect ive and o ptimi zed appli cati o n of
the method 64 . Free ce ll s can prov ide valu abl e
in fo rm ati on in labo rato ry ex perime ntati o n but have
65
li mi tati o ns in indu stri a l appli catio ns . The use of
free ly sus pended mi crobi a l bi o mass suffe rs with
d isad vantages li ke sma ll partic le s ize, low mechani ca l
strength and di fficulty in seques tering bi o mass and
e fflu ent. The immo bili zed bi oadsorbe nt bi o mass
pac ked in sorpti on co lumns, w hich are effecti ve fo r
continu ous remova l of heavy metals can ope rate o n
cycl es consisting of loading, regenerati o n and
rin sing 66 . Ibanez and U mets u67 have de mo nstrated the
ability of proto nated al g in ate beads in the re moval of
chro mium , copper, zinc, ni ckel and co balt io ns from
dilute aqu eous soluti o ns. Bio mass immo bili zed in a
range of inert materi als like, sil ica, po lyacry lami de,
po ly meth ane and po lysul fo ne has been used in a
vari ety of bi o reacto r co nfi g urati o ns, inc ludin g
rotating bi o log ical co ntracto rs, f ixed reacto rs, tri c kl e
filters, flu idi zed beds and a ir li ft bi o reacto rs 65 . Karn a
68
et aL. immo bili zed Phormidium va lde rianum BD U
3050 ] in po lyv in y l foa m a nd used fo r the re mova l of
C a-?+, C 0 2+, C u-?+ a nd N 1-'?+ . As hta na et al. 63 repo rted
th at Ca-alg in ate immob ili z in g age nt is very effecti ve
in ni cke l biosorpti o n and used in o rgani c sa lts like
NaC I and Ca (N03)2 fo r desorpti o n of ni cke l fro m
immo bili zed mi crob ia l bi o mass. HC I a nd EDTA are
94 1
the most effic ient desorpti o n agents fo r ni cke l
re movaI62 ,68.
Plant-microbe interaction in metal remediation
Heavy metal tox ic ity to pl ants can be reduced by
the use of pl ant g row th promoting bacteri a, free-li ving
soil bacteri a that e xert so me benefi c ial effects on
pl ant develo pme nt whe n they are appli ed to seeds o r
70
incorpo rated to seeds . Pl ants ca n acce lerate
bi o remedi ati o n in surface so il s by the ir abil ity to
stimul ate so il mi croorgani sms thro ug h th e re lease of
nutri ents fro m the soil and transpo rt of oxygen to the
7o
rhi zosphere . Pl ant g rowth pro moting bacteri a that
contain ACC deamin ase may act to in sure th at the
eth y le ne leve l does not impa ir root growth and
facilitate the fo rmation of larger roots, whi ch enhance
71 72
seedling surviva l . Burd et al. reported that a metal-
res istant soil bacterium Kluyvera asco rbata S UD 165
pro moted the g rowth of cano la (Brassica campestris)
in the presence of hi g h concentratio ns of nickel and
the increase in growth rate is du e to the abi lity of the
bacterium to lo wer the leve l of eth y le ne stress in the
seedlin gs. They furth er iso lated siderophore over
produ c ing mutants, K. ascorbata 165/26 whi ch was
able to prov ide suffic ie nt io ns fo r the g rowth of Indi an
mu stard (Brassica j uncea) and to mato (Lycopersicon
esculentum) in the presence of hi g h concentrati o ns of
N i, Pb, and Zn . Studies of Bu rd et aL. 73 s uggested the
best method to preve nt plants fro m beco ming
chl oroti c in the presence of hi g h le ve ls of heavy
metals was to provide th e m with a siderophore-
produc ing bacterium , Kluyvera ascorbata S UD 165.
M ycorrhi zal fun g i a lso reduce metal tox ic ity to the ir
host pl ant by bind!ng heavy meta ls to th eir ce ll wall
o r surro undin g po ly saccharides. Binding sites w ithin
cell s of ecto mycorrhi zal fung i are metall othi onein-
like prote in s, w hi ch have hi g h cys tine conte nt and
7o
perhaps po ly phosphate g ranul es .
Field applications
The mos t impo rtant bi o tec hn o log ica l application of
meta l-mi c robe interacti o n is in bi o leaching,
bi o re medi atio n of po lluted sites and min era li zati on of
po lluting o rg nin c matter l6 l) ranium is extracted fro m
uranite o re th ro ugh bi o leac hing by indirect acti o n
w ith T.Jerroxidans . In thi s case, ferri c iro n in aci di c
so luti o n, w hi c h th e orga ni sm gene rate w he n ox id izin g
py rites (FeS2), ox idi zes in so lu ble UO to so lu ble
74
UO/+ . T he ex tracti o n of meta ls s uch as Co, Mo , Ni,
Pb, and Z n fro m sul fid ie o res by bi o leaching is
technbicall y feas ibl e. Gencor (South Africa) is cun'e ntly
942 INDIAN J EXP BIOL, SEPTEMBER 2003
developing tlie BioNIC process to recover nickel from
low-g rade sulfide ores 75 . 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 Uganda 76 .
Various microbiall y reducible metals, especially ferric
iron in complexed form to keep it soluble at circum
neutral pH, can be used as terminal electron acce ptors
in in situ anaerobic bioremedi ation of sites polluted
with tox ic organi cs 77 . It is possible to bi oremedi ate in
situ sites polluted with chromate or dichromate [Cr
(Vl)] by stimul atin g the reducti on of th e Cr (V I) to Cr
(Ill) by bacteri a25 . Fungi ca n co nve rt oxidized
selenium to volatite-methylated selenides for escape
into th e atmosphere, a removal by volatilization 78
Beseker79 reported Penicillium fu.niculasul1l was ab le
to ex tract more than 50% Ni and 75 % Zn from test
solu tions containin g 100 mg rt of metal at pH 6.6 and
6. 5 respectively. A poten t algal biosorbent Alga
SorbT .Y1 developed using a fresh water alga Ch Iarella
vu.lgaris and AMT-BIOCLAIMTM (MRA ) developed
using Bacillus biomass are used to treat wastewater
and metal recovery respecti vel/ 6 .
Future prospects
Microbes ca n significantly affect th e di stribution of
metals in th e environment, since th ey have developed
means to use them for their benefit. This clearl y holds
promise for effecti ve, eco nomi ca l and eco-fri endl y
metal bioremediation tec hnol ogy for indu stri al
ex ploitati on and pollution free environm ent. A good
and efficient metal biosorbent ca n repl ace the
co mm ercial ion exchange res in s that have been used
co nvent ionally for metal removal. However, th e bas ic
knowl edge of microbi al metal bi oremediation
mechani sm is inev itabl e for the development of
commerciall y viable potent bioso rbent. Altho ugh
se vera l mi crobi al metal bioremedi at ion approaches
are establi shed to combat heavy metal polluti on from
anthropogeni c and natural so urces, none is yet in wide
spread use. Innovative, eco nomi call y fea sible and
novel biomass regenerati on and conversion of the
recovered metal into usabl e form are th e best options
to attract more usage of bi oso rbents. It is time to
II1ttlate more comprehensive interd isciplinary
approach between biotechnologists and metallurgists
to bring lab sca le bi oremedi ation process to land scale
tec hnology that will be accep tabl e for industriali sts.
Acknowledgement
One of the authors CPR) is indebted to UGC, New
Delhi fo r th e award of Teac her fell ows hip for doc toral
studies and to the Management of Vivekananda
College, Thiruvedagam, Madurai for deputation under
F.I.P. program.
References
I Ceribasi I H & Yeti s U, Biosorpti on of Ni (ii ) and Pb (ii) by
Phanaerochaete chrysospo riul1I from a binary metal stystem -
kin eti cs, Wate r SA ,24 I (200 I) 15.
2 Hernandez A, Mellado R P & Martin ez J L, Mctal acc umul at ion
and va nadi um-induced multidrug resistance by environmclllal
isolatcs of Escherichia herdrna llni and Elllerobacter cloacae.
Appl Enviroll Microbiol. 64 ( 1998) 43 17.
3 Tyagi P, Budd hi D, Chod hary R & Saw heny RL, Degradati on
of ground wa ter quality due to heav y metals in industri al arcas
of India- A review, lJ EP, 20 ( 1999) 174.
4 Mudakavi J P & Naraya na B V, Toxic heavy metal
co ntamin ati on of th e soil and biota: Part II- Environmental
imp li cati ons, IJ EP, 18 (2) ( 1997) 108.
5 Eislcr R, Nickel hazards to fi sh wild life and in vertebrates: A
sy noptic review, in COlllaminalll hazard reviews, 1-35, Patllxtent
Wild Life Research Centre, Lau reL. Mat )'land. 2000, I.
6 Co lbou rn P & Thronton , Lead pollution in ag ricullUral soil s, J
Soil Sci. 29 ( 1978) 5 13.
7 Roa ne T M & Pepper I L, Microorgani sms and metal
polluti on, in Ellvirollmelltal Mi crobiology, cdited by Maier R
M, Pe pper I L & Gerba. C B (Academ ic Press, Lo ndo n, NW I
7B Y.U K) 2000, 55.
8 Salol11ons W & Forstn er U, Tracc mctal analysis o n polluted
sedi me nts. Pa rt 2. Evalua tio n of envi ronmental im pac t,
Ell viron Techllol Lett, I ( 1980) 517.
9 Lippard S J, Mctal s in medicinc, in Bioinorgallic chemistry,
edi ted by Bertini!. Gray H B, Lippard S J & Valantine J S.
(Pub. Viva Book Pvt, New Delhi ) 1998 , 506.
10 Ol so n J W, Mehta N S & Maicr R J, Requ ire me nt of ni ckel
mctabo li sm proteins HypA and HypB fo r full ac tivity of both
hydroge nase and urcase in Helicobacter pylori. Mol
Microbiol, 39 (200 I) 176.
II Va n Assc he F & Chys ters, Effccts or metal s on enzy me
activity in pl ants, Plant Cell En viron, 13 ( 1990) 195.
12 Mai er R, Lipid lather remo ves metal s, En viron Heal Per. 108
(2000) 320.
13 ATSD R- 200 I, CERCLA Pri orit y Li st of Hazardous
Substances, (cI http:// WWW.atsdr. cc/c.gov/ C/ist.htllll).
14 Trivedi R P & Gundap Raj. A slUd y of industri al water
pollution, In Encyclopa edia oj en virollmelltal sciences
(Akashdeep Pu bli shing House, New Delhi ), 1992 , 185.
15 Sposito F G, The chemistry of soil s. in Ellvironlll elltal
M icrob iology, ed ited by Maicr R M, Pepper I L & Ge rbil C B,
(Academic press, London , NW I 7BY.U K) 2000, 406.
16 Ehrlich H L, Microbes and metals, Appl Microbiol
Biotechnol, 48 ( 1997) 687.
17 Xian X, EITect of chemi ca l fo rms of cad miulll zinc and lead in
polluted soil s and th eir uptake by cabbage pl ants, Plallt Soil,
11 3 ( 1989) 257.
18 Lena Q M & Rao G N, Heavy metals in th e environment , J
Ellviron Qual. 26 ( 1997) 264.
19 Kri shnamurthi G S R, Spec iat ion of heavy meta ls: An
approac h for re med iati on of co ntaminated soil s, III
Remediation engin eering oj cOlltamillatec! soils . edited by
Wi se D L, Trantalo D J, Cichon E J, Inya ng H I &
Stottmeister U (Marcel Dekker Inc Ne w York ) 2000, 709.
 RA1ENDRAN et al. : MICROBES IN HEAVY METAL REMEDIATION
20 Sauerback D R & Rietz E Soil chemical evaluation of
different ex tractants for heavy metals in soil s, in Comm:
Europe Communiites Report EUR 8022. En viron. Eff Org
Inorg Contam Sewage Sludge , CA 99: 193726 ( 1983) 147.
2 1 Ri chter 0 R & Theis R L. Nickel speciation in a soil/water
system, in Nickel in environment, edited by Nrigu, J 0, (New
York, Chichester, Brisbance, Toronto, John Willey & sons)
1980, 189.
22 Beveridge T J & Doyle R, Metal ions and bac teri a, in
Microbes and metals, Mini-review by Ehrlich H L, Appl.
Microbiol Biotechnol, 48 (1997) , 687.
23 Summers A P & Sugarman L I, Cell fre e mercury ( II )
reducing ac ti vi ty in a plasmid-bearin g strain of Escg heriachia
coli , 1. Bacte riol, 11 9 ( 1974) 242.
24 Summers A P & Si ver S, Mi crobi al transform ations of metals,
Annu Rev Microbiol, 32 ( 1978) 637.
25 Wang Y T & Shen H, Bacterial red uction of hexava lent
chromium, J In d Microbiol 14 ( 1995) 159.
26 Mcas ki e L D, Dean A L R, Cheth am A K, Jakeman R J B &
Skarnu li s A J, Cad mum acc umul ati on by Citrobacter spp: the
chemi cal nature of the accumul ated metal prccipitate and its
locati on in bacterial cell s. J C en Microbio/, 133 (1987) 539.
27 Ferris F G, Schultze S, Wriller T C, Fyte W S & Beveridge T
J, Metal interacti ons with microbial bi ofilms in ac idic and
neutral pH environments, App/ Envrion Microbiol, 55 ( 1989)
1249 .
28 Pazirandeh M, Wcll s B M & Ryan R L, Development of
bacterial based heavy metal biosorbcnts: enhanced uptake of
cadmium and mercury by Escherichia coli expressing a metal
binding motiff, Appl Environ Mi crobiol, 64 ( 1998) 4068.
29 Gadd G M, Microbial contro l of heavy metal pollution, in
Microbial control of poilution edited by Fry J C, Gadd, G M,
Herbert R A, Jones C W & Watson-Graik bridge, (LA.
Cambridge Universit y Press) 1992,59.
30 Lloyd J R & Lovley D R, Microbi al detox ification of metals
and radi onu clides, Cu rr Opi Biotech, 12 (2001 ) 253.
3 1 Gadd G M, Bioremedial potenti al of metal mobilizati on and
immobili zati on, CLirr Opi Biotechnol, II (2000) 27 1.
32 Blanco A. Immobilizati on of nonviab le cya nobacteri a and
their use for heavy metal adsorption from wa ter in
En vironmental biotechnology and cleall er bioprocesses,
edited by Oluguin E J, Sanchez & Hern andez E (Philadelphi a,
Taylor & Amp, Francis) 2000, 135.
33 Wase J & Forster C, in Biosorbellls for metals ions, (Bri stol)
1997, 1.
34 Alexander M, Bi oremed iati on of metal and other pollutants, in
Biodeg radation and biorelll ediation, Second edi ti on,
(Acade mic Press, New York ) 1999 , 377.
35 Torrens J L, Herman D C & Miller-Maier R M, Biosur fac tants
(rhomphn olipid) sorpti on and impac t on rh omphnoli pid-
facilitated fr moval of cadmi um from vari ous soils under
saturated !low conditi ons, Enviroll Sci Techllol, 32 ( 1998)
776.
36 Sand W, Rohde K, Sabotke B & Zenneck C, Eva luati on of
LeptospirillLlIll f erroxidalls for leaching. Appl En viron
Microbiol, 58 ( 1992) 85.
37 Joerge r T K, Joerger R, Olsson E & Granqvi st C G, Bac teri a
as worke rs in th e li ving factory: metal accumul atin g-bac teri a
and th eir potenti al fo r materi al sc icnce, Tibt ech 19 (200 I) 15.
38 Ochi ai E, Bioinorganic chemi stry: An introduction (A lign and
Bacon, Boston) 1997, I.
943
39 Nealson K H Rosson R A & Mayers C R, Microbial redu cti on
of manga nese and iron: New approaches to carbon cycling,
App Environ Microbiol, 58 ( 1992) 439.
40 Tomsetl A B, Sewell A K, Jones S J, Miranda J R & Thurman
D A, Metal binding proteins and metal regulated gene
expression in hi gher plants, In Inducible plant proteins, Soc.
Exp. BioI. Sem. Series, edited by Wary J L, (Cambridge
University Press. New York) 1992, I. .
41 Mej are & Bullow L, Metal- Binding proteins and peptides in
bioremed iati on and ph ytoremediation of heavy metals, Trends
Biotech, 19 (2001 ) 73.
42 Lichtlen P & Schaffner W, PUlling its fin gers on stressful
situati ons: th e heavy metal reg ulatory transcripti on facto r
MTF- I, Bioessays 23 (200 I) 10 IO.
43 Gupta A, Morby A P, Turner J. S, Whitton B A & Robinson
N J, Deleti on within the metallothi onein locus' of Cadmium
tolerant Synechococcus PCC 630 1 involving a hi ghl y iterated
palindrome (HIPI ), Mo Microbiol 7, ( 1993) 189. .
44 Cervantes C, Ji G, Rimirex J L & Silva S, Resistance to
arsenic compounds in microorga ni sms, FEMS Microbial Rev.
15 ( 1994) 355 .
45 Silver S & Phung LT, Bacterial heavy metal resistance: New
surpri ses, Anl1 Rev Microbiol. 50 ( 1996) 753 .
46 Schmidt T & Schlegel H G, Combined nickel-cobalt-cadm ium
resistances encoded by th e ncc locus of Alcaligenes
xylosoxidalls 3 1A, J Bacteriol, 176 ( 1994) 7045 .
47 Mergeay M, Towards an understandin g of the ge netics of
bacteri al metal resistance, Tibtech, 9 ( 199 1) 24.
48 Tagha vi S, Delanghe H , Lodewyckx C, Mergeay M & Lelli e
D V, Nickel- resistance- based minitransposons: new tools fo r
ge neti c manipulati on of environmental bacteria, Appl Ell viron
Microbiol, 67 (200 1) 1015.
49 Chen S & Wil son D B, Construction and chracteri za ti on of
Escherchia coli ge netically engineered for bioremediatio n of
Hg2+ contaminated environments, Appl Environ Microbiol, 63
( 1997) 2442.
50 Kri shnaswa my R & Wil son D B, Construction and
characterization of an E. coli strain ge neti ca ll y engineered for
Ni (II) bioaccumulation , Appl En viron Mi crobiol, 66 (2000)
5386.
5 1 Bang S W, Clark D S & Keasl ing, J D, Enginerrin g hydroge n
sulfide producti on and cad mium removal by expression of the
thi os ulfate redu ctase gene (phs ABC ) from Salmonell a
enteri ca serovar typhimurium in Escherichia coli. Appl
Envion Microbiol, 66 (2000) 3939.
52 Vall s, M, Atri an, S, Loveno, V D & and Fernandez L A,
Engi neerin g a mouse metallothionum on the cell surface of
Ralsatonia elllropha CH 34 for immobili zati on of heavy
metals in soil , Na t Biotecilllol, 18 (2000) 66 1.
53 Kotrba P, Pos isil P, Lorenzo V D & Ruml T, Enhanced
metalloprotein of E.coli cell s due to surface di spl ay of beta
and alpha-domains o f mammali an metall othi onein as a fusio n
to Lam B protein , J Receptor Sigllal Transduct Res, 19 ( 1999)
703.
S4 Samuel son P, Wern erus H, Svedberg M & Stahl S,
Staph ylococcal surface di spl ay of metal-bind ing polyhistydiyl
peptides, Appl Ellviron Microbiol, 66 (2000) 1092 .
55 Watanabe K, Microorganis ms relevant to bioremedi at ion.
Curr OpiBio Tech,12(2001)237.
S6 Ahuj a V, Vo hra, P K, Kas hyap D R & Tewari R, Adsorpti on
of heavy metals (Pb 2+ and Cd 2+) by free and immobi li zed
biomass of Acil/('[obacter allit/"{/IIIS, IlM, 4 1 (200 I) 279.
944 INDIAN J EXP BIOL, SEPTEMBER 2003
57 Vasudevan p, Padmava th y V, Tewari N & Dhingra SC ,
Biosorpti on of heavy metal ions. J Sci In dus Res, 60 (200 I)
120.
5S Ei izabcth K M & Anuradha, T V R, Bi osorpti on of hexava lent
chromium by non-pathogeni c bacteri al cell prepa rati ons, UM,
40 (2000) 263.
59 Rajendran P, Ashokkumar B, Muthukri shnan & pl ant eth ylene concentrati ons by pl ant grow th pro motin g
Gun asekaran P, Tox icity assessment of ni cke l using
Asperg illus niger and it's removal rrom an industri al efflu ent,
Appl Biochem Biolechnol 102 (2002) 20 I .
60 Volesky B, Detox ificati on of metal- bearing effluents: biosorpti on
for the next century, Hydromelallurgy, 59 (200 I) 2 16.
6 1 Fourest E & Roux J C, Heavy metal biosorption by fun gal
myccli al by products: mechani sms and in n uence of pH , Appl
Microbiol Biolechnol. 37 ( 1992) 399.
62 Ramteke P W, Bi osorpti on of Nickel (II ) by Pseudomollas
sllil ze ri. J En viron Bioi. 2 1 (2000) 2 19 . .
63 Asth ana R K, Chatterj ee S & Singh P, In vesti ga ti ons on ni ckel
biosorpti on and its remobilizati on, Proc Biochelll. I ( 1995 )
729.
64 Volcsky B, Biosorpti on for the nex t century, Illlerncuional
Biollleiallurgy Symposium . EI. Escori al, Spain , ( 1999) 20.
65 Gadd G M & White C, Microbi al treatment of metal
polluti on - A wo rk ing biotec hnology ? T1B TECH. II (1993 )
353.
66 Gupta R. Ahuj a P, Kh an S, Saxena R K & Mohapatra H,
Microbial biosorbcnt s: Meetin g chall enges of heavy metal
pollu tion in aqu cous solution, Curl' Sci. 78 (2000) 967.
67 Ibanez J P & Umctsu V, Potential of protonated alginate
beads for heavy metals uptake, Hydrollleiallurgy, 64 (2)
(2002) 89.
68 Kama R R, Uma L, Subramani an G & Mohan P M.
Biosorpti on of tox ic mctal ions by alk ali extrac ted biomass of
marin e Cya nobac terium.PhorJll idiUlII I'a lderialllllli BDU
3050 I. Wo rld J Microbiol Biolech. IS ( 1999) 353.
69 Kl opper J W, Li fshitz R & Za blotowicz R M, Frcc li vin g
bacteri al inoc ul a for enhancing crop prod uctivity, Trellds
Biolechnol. 7 ( 1989) 39.
70 Wenze l W W, Manipul atin g rhi zos phere chemistry to cont rol
metal and orga ni c contaminant avail ab ility and implicat ions to
Phytoremedi ati on. in 2nd Imernalional conferell ce Oil.
conlaminenls ill soil en vironlllent in Ih e A uslralia - Paciji'c
reg ion. New Delh i, ( 1999) 12.
7 1 Gli ck B R, Penrose B R & Li J, A model fo r the lowcri ng of
bac teri a, J Th eor Bioi. 190 ( 1998) 63.
72 Burd G I, Di xo n G & Gli ck B R, A pl ant grow th -pro moti ng
Rhizobaclerium th at decreases nickel tox icity in seedl ings,
Appl En vi ron M icrobiol. 64 ( 1998) 3663.
73 Burd G J, Di xo n D & Gli ck B R, Pl ant growth-promoli ng
bac teri a th at decrease heavy metal tox icity in pl ants, Call J
Microbiol. 46 (2000) 245.
74 McCready R G L & Gould W D, Bi oleaching or uraniu m, in
Microbia /mineral recovery, edited by Ehrli ch H L & Brierl ey
C L, (M cGraw- Hili , New York ) 1990, 107.
75 Dew D W & Miller D M, The BIONI C process. Bioleaching
or mineral s sul fide concentrates for recovery or nickel, in
Conference p roceedin gs. Int ernalioll al Biohydrom elall rgy
Symposillm IBS97 BIOMINE97, (Austra li an Mi neral
Form ati on Gleenside, South Australi a) 1997, M. 7.1 .1 .
76 Briggs A P & Mill ard M, Cobalt recovery using bacteri al
leaching at th e Kasese Project Uga nda, in COllferell ce
p roceedings. Inlernalional Biohyd romelallrgy SYIIIPosiulII.
IBS97.BIOMINE9 7 (Australi an Mineral Formati on.
Gleenside, South Australi a) 1997 , M. 2.4. 1.
77 Lovely D R, Di simul atory metal redu cti on, Anl1u Rev
Mi crobiol, 47 ( 1993) 263.
78 Frankenberger W T & Karl son U, Di ssipati on of soil seleni um
by mi crobial vo latili zati on, in Biogeochemistry of lra ce
I'Ilelals. edited by DC Adri ano Lew is, (Boca Rati on. Fla)
1992 , 365.
79 Bosecker K, Biosorpti on of heavy metals by ril amenlous
fun gi, in Biohydrollleial/urg ical leclllw logies. Vol-II. The
minerals. mela/s and malerials sociely. edited by Torm a, A E,
Apel M L & Brierl ey C L (Warrendale, Pal 1993 , 55.