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
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
ID
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

INDIAN J EXP BIOL, SEPTEMBER 2003

936

their pathological effects, whi ch pose health hazard to

envi ronment (T abl e I ) 14.

Metal availability
5
Sposito'
categori sed me tal s as bioavail abl e
(prec ipitated, non- sorbed and mobil e) and nonbioavailable (precipitated , sorbed , and non-mobile)
metals. In nature , metals and Il!etalloids ex ist mostl y
as catio ns, oxy anion s, or both in aqueoll s soluti o n and
mostl y as salts or oxides in crystalline (minera l) form
7
o r as amorph ous prec ipitates in solubl e form . ' 6 . The
mobility of metals as hydrated io nic salts is depe ndent
on two factors 1. The meta llic e le ment that is
parti c ipating as positi vely charged io ns (cati o ns) and
2. The one, which makes up negati vely charged
compo nent o f the salt. The cati o nic/a ni o nic so lubility
re lati ons hip is in T abl e 2 . Geoche mi cal fo rms o f
heavy metals in soil affect th e ir so lubility , which
direct ly influence the ir bi oavail abili ty ' 7. Le na and
l8
Rao repo rted mo bility and bioava il ability of metals
in so il s is in the orde r of Zn > Cu > Cd > Ni , based on
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
chem ical form o f the metal even thou g h the
enviro nmental and edaphi c conditi o ns s uch as pH ,
redo x statu s, and so il o rgani c matte r conte nt have
sig ni ficant influence ' '! . Studi es of Sa uerbeck and
Ri etz 20 have indicated the ex iste nce of different
bi nding fo rms and strong ly p H depenent so lubility
effec ts of the trace e le ments. Soil cation exchange
capacity (CEC) affect meta l bi oavail ibilty and it
depends o n orga ni c matter and cl ay co nte nt o f the
so il. T he tox ic ity of metals within so ils with hi g h
CEC is low even at hi gh to tal me ta l co ncentrati o n and
7
in contrast the toxic ity is vice-versa in lo w CEC .
Under ox idi zed o r aero bi c co nd iti o n (+ 800 to mY )
"metals are usua ll y fo und as so lubl e cati o ni c fo rm s

2
(Cu +, Cd 2+, Pb 2+ & Ca2+) and in reduced or a naerobi c
conditi o ns (0 to -400 mv) they are fo und as CuS ,
7
PbS , and CdC03, precipitates . At low soil pH, the
me ta l bioavailability increases due to its free ioni c
spec ies and in contrast, hig h so il pH dec reases due to
in so luble metal minera l phosph ate a nd carbo nate
form ation . Ho we ver, in contrast to other metal
bioavailabilities, at hi g h pH range o f 5-9, ni c ke l may
be adsorbed o n iron a nd manganese ox ides, o r fo rm
compl exes with inorganic li gands (OH -, S042- , cr o r
NH3)21.

Metal microbe interaction

Microbi al interacti o ns w ith heavy meta ls were
rev iewed by, Ehrli ch ' 6 and Beveridge a nd Doy le 22 .
Mi crobi al interacti o ns with sma ll qu antiti es of meta ls
o r metalloids do not exert a maj or impact o n metal o r
metalloid d istributi o n in the e nviro nme nt, whereas
interactions with larger quantiti es are required in
energy metabo li sm, fo r in stance, to ha ve no ti ceabl e
impac t ' 6 . Howe ver, due to stron g io ni c nature metals
Table 2 - Summary of cati on/a ni on solu bi li lY relati onshi p of
metals
Anions

Catio ns

All ani ons

Na+, K+, NH/

All metals

Solub le
Solub le

All metals except

Pb 2+ , Ag+, Hg2+

Solu ble

SO ~ -

All metals except

Ba 2+, Sr2+, Pb2+

Solub le

0 2-, S2-, OW

All metals except

Ca 2+, Ba 2+, Sr2+
All metalli c salts except
Na+, K+, NH/

Insolu ble

NO ;- , NO -2 ' C 2H30 2,
Mn0 4, CIO ~ , CIO ~
CI- , Br- , 1-

CO ~ - , PO ~- , SO ~BO j- , F , SiO j-

Solu bil ity

Inso lub le

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

Metal
Hg

Use

Effect

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
Cr

Ferti I ize r, pl astic, pigment

Tannin g, paints, pigmenl , fun gicide

Zn

Co

Fertil izer
Vilam in B- J2

Se
Ni

Coa l, sulfur
Electro plating

Kidn ey damage, inju ry in cns and mental retardati on

Nephritic, cancer and ulcerati on
Vom iting, renal damage and cra mps
Diarrh oea. low blood press ure and paralys is
Damage of li ver, kidney , spl een and nervo usness
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
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.
28
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 ysico29
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.

INDI AN J EXP BIO l , SEPTE MB ER 2003

938
Mechanisms of metal tolerance

spec ific actI vIty. Mi croorgani sms can affec t heavy

metal concentrati o ns in the e nvironment bec au se they
ex hibit a strong ability fo r metal removal from
so lution ; thi s can be achieved th roug h e ither
enzy matic o r non-enzymatic mechanj sms39 . A voidance,
restricti o n of meta l entry into the ce ll, e ither by
reduced uptake/acti ve efflu x o r by th e fo rmati o n of
co mplexes o utside the ce ll a nd sequestrati o n,
reducti o n of free io ns in the cy toso l e ither by
sy nthes is of li gands to achieve intrace llu lar che lati o n
or by co mpatme nta li zati o n are th e two major
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
meta l tolerance in mi cro bes are shown in Table 4.

O rgani sms respo nd to heavy metal stress using

di ffe rent defense systems (Fig. 1) such as exc lus io n,
compartmenta li zati on, fo rmati o n of co mplexes and
sy nthesis of binding prote in s like metallo thione ins
38
(MTs) and phytoche latin s (PCs). Ochari has di vided
general tox ic ity mechani sm fo r meta l io ns into three
categories, 1. blocking the essenti a l bi olog ical
funct io nal groups o f bi o mo lecul es espec ia ll y pro te in s
and e nzy mes, 2. di spl ac ing th e essenti al meta l ion in
bi o mo lec ul es
and
3.
modifyin g
the
acti ve
conformati o n of bi omo lec ul es res ul tin g th e loss of
Table 3 - Exam ple of microorgani sms th at take up heavy metals
Microorgan isms

Elemen ts

Uptake

Metallothioneine

(% dry wt)
Zooglea spp.
Citrobacte r spp
Ba cillus spp
Chlorella vulga ris
Rhizopus arrh iZ.lIs

Aspergillus niger

Co
Ni
Cd
U
Cu
Zn
Au
P
Ag
Hg
Th

Meta ll othi o ne ines, di scovered abo ut 45 years ago,

pl aya centra l ro le in heavy meta l me tabo li sm and in
the management of vari ous forms of stress.
Meta llo thi o nein es (MTs) are lo w molec ul ar weight
(6-7 kDa), cystine- ri c h prote in s, di vided into three
di ffe re nt cl asses o n the bas is of the ir cystine content
and s tru~ture . The C ys-Cys, Cys-X-Cys and C ys-XX-Cys, mo tifs (in whi ch X de notes any amino acid )
41
are characteristi c a nd inv ari ant fo r metall o thi o ne ine .
42
Studi es of Li chtl e n and Schaffner
show meta l

25
13
170
900
15
14
10
10
54
58
19

Protein denaturation
(Hg, Pb & Cd ).

Inhibition of cell division

(Pb. Cd. Hg & Ni)

/
MIC ROB ES

Inhibiti on of enzyme acti vity ]

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

Cell membra ne disr uption

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

Transcription inhibition(Hg)

~
DNA - . mRN A - . Protein synt hes is

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

t
Translation inhibition
(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 +

Cd 2+-& Z n2+

Anion effl ux (ATPase)

Efflux (AT P ase)
Reducti on
Efflux
Decreased uptake
Cat io n effl ux
Decreased uptake
DNA damage.

Hg2+
Co 2+ & Ni2+

Cr0 42.
Cd2+ Cd2+ & Zn2+

cr02 .
Cu 2+

regul atory transcriptional factor-l (MTF-l ) was

essenti al for basal and heavy metal induced
transc ripti on of the stress- responsive metallothioneinI 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 Grampositive bacteri a lack the ArsA protei n44.

- 1

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 +,
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 protonanti 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-transferase 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

INDI AN J EXP BI OL, SEPTEMBE R 2003

940

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,
in
po lysaccharides,
parti c ipate
in
hydroxy l
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
(Fu nga l sp p)

Effl ux pump
Passive mec hanism

(A.e lllrophu s)

Ac ti ve mecha nism

Ex tra ce ll ular complexati o n

(CilrobaCl er spp)

Extra cellular polymeric substance

(S.a ure ll s)

Ion exchange reactio n

(S.pia l ellSis)

Complexa tio n with oxygen and

nitrogen ligands (P. pUl ida)
Polysacc harides (Z rallligera)
Proteins (A. bisporous)
Nuc leic acid

Destruction of co mp lexing
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 o31
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
'?+ . As hta na et al. 63 repo rted
C a-?+, C 0 2+, C u-?+ a nd N 1th 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

the most effic ient

re movaI62 ,68.

94 1

desorpti o n agents fo r ni cke l

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 metalres 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 siderophoreproduc 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 oneinlike 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.
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