microbes in heavy metal remediation ijeb 41(9) 935-944

7/26/2019 Microbes in Heavy Metal Remediation IJEB 41(9) 935-944

1/10

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

li

cation for metal

accumulation/detoxification. This article summarizes the potentials

of

microbes in metal remediation .

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 wastes

3

,

automobile

ernissions

4

, mining activit/,

and

agricultural

practices

6

. 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

Pepper

7 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 metal s (Ag,

Cd

,

Sn

, Au,

Hg, Ti , Pb, AI , and metalloids Ge,

As

,

Sb

and Se) ,

and

(3) th

e nonessential ,

nontoxic

metals with no

known biological effects (Rb, Cs,

Sr

, and T).

Ba

sed

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

*

Cor

respondenl aUlhor :

Phone: 0452-5456478; 452-458209

Fax 0452-545 8478; 0452-459139

Emai l pgun

a@e

lh.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

en

zyme

catalysis, nutrient transport, protein structure,

charge

neutraliziation

and contro

l

of

osmatic

pressure

9

. Olson

et al

I

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 rnicronutrient s 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

prese

nt 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 Sub

stances 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 .

Chan

ges

in trace

element

profile of the soil causes

physiological and genetic

changes

in various life,

such as plants, aquatic

and

benthic fauna, insec ts,

earthw

orms, 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

permis

sible limit of

World H

ea

lth

Organization

.

They

have

also reported

that the

se

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

metals w ith


2/10

936

INDIAN J EXP BIOL, SEPTEMBER 2003

their pathological ef fects, which pose health hazard to

en

vi

ronment (Table

I )

1

4.

etal availability

Sp

os ito

5

ca tego ri

se

d metals as bioava il able

(prec ipitated, non- s

orb

ed and mobile) and non

bioavailable (precipitated , so rbed , and non-mobile)

metals. In nature, metals a

nd

Il etalloids

ex

ist

mo

s

tl

y

as ca tio ns,

oxy

anions, or

both

in

aqueo ll

s solution

and

mostl y as

sa

lts or

oxides

in

cry

stalline (min eral) form

or as amorphous prec ipitates in so luble form

7

.

' 6

. The

mobility of metals as hydrated ionic sa lts is depe ndent

on two factors

1

The meta llic e lement that is

participating as positi

ve

ly charge d io ns (cations) and

2.

Th

e one,

which makes

up n

ega

ti vely charged

c

omp

onent o f the

sa

lt. The cati onic/ani onic

so

lubility

rela ti onship is in T abl e 2 . Geochemica l fo

rm

s of

heavy metals in soil a

ff

ect their

so

lubility ,

which

di r

ect ly influen

ce

the ir bi

oava

il

abili ty

'

7.

Lena a

nd

Rao

l8

reported mobility and bio

ava il

ability

of

metals

in so il s is in the order of Zn > Cu > Cd > Ni , based on

their solubility and ge

oc

hemica l form s. Th e fate of

tox ic meta ls

in

so

il

s depend s mainly on the initial

chem ica l form of the metal

eve

n though the

enviro

nment

al and edaphi c

co

nditions such as

pH

redox status, and

so il

orga nic matte r co ntent have

signi fica nt influen

ce

'

' . Studi es of

Sa

uerbec k and

Rietz

20

have indica ted the ex istence of different

bindin g fo rms and stro ng ly p H depenent

so

lubility

effec ts of the trace e lements. Soil ca tion exchan

ge

capaci ty

(CEC)

af fect metal bi

oava

il ibilty and it

depends on orga

ni

c matter and cl ay co ntent of the

so il. The tox ic ity of metals within

so

ils with hi gh

CEC is l

ow

even at high total meta l

co

n

ce

ntra ti on and

in

co

ntras t the toxic ity is vice -ve rsa in low CEC

7

.

Un

de

r ox idized or

ae

robic

co

ndition

(+ 800

to

a

mY )

"metals

are

usua

ll

y found as

so

luble ca ti oni c fo rms

(Cu

2

,

Cd

2

+, Pb

2

+ Ca

2

+)

and

in redu ced or a nae robic

conditions (0 to -400 mv) they are fo und as CuS ,

PbS ,

and

CdC0

3

, prec ipitates

7

. At low soil pH the

metal bioavailability increases du e to its free ionic

spec i

es and

in contras t, high

so

il

pH

dec r

eases

du e to

inso luble metal minera l ph os ph ate and

ca

rbonate

form ation . However, in co ntrast to

oth

er

met

al

bioavailabiliti es, at high pH range of 5-9, ni cke l may

be adso rbed on iron and manga nese ox ides, o r fo rm

compl

ex

es

with

inor

ga nic li ga nds (

OH

-,

S042-

,

cr

or

NH

3)21.

etal microbe interaction

Microbial interac ti ons with heavy me ta ls we re

rev iewed by, Ehrlich

' 6

and Bev eridge and D

oy

le

22

.

Mi crobial interacti o ns with sma ll qu antiti es of meta ls

or metalloids do not

exe

rt a maj or

imp

act on m

eta

l o r

metalloid d istribution in the environment, whereas

inter

ac

tions with lar

ge

r

qu

antiti

es

are re

quir

ed in

ener

gy met

abo li sm, fo r

in

stan

ce

, to have no ti cea

bl

e

impac t

' 6

.

How

ever,

du

e to strong ionic na

tur

e metals

Tab le 2 - Summary of cati on/ani on solu bi li lYre la tionshi p of

metals

Anions

Catio ns

So lu bil ity

A

ll

ani ons

Na ,

K

,

NH/

Sol

ub

le

NO

;-

, NO 2 ' C

2

H

3

0

2

,

A ll metals

Sol

ub

le

Mn0 4, CIO , CIO

CI- , Br- ,

1-

A

ll

metals except Solu ble

Pb

2

+,

Ag , Hg2

SO A

ll

metals except Solub le

Ba

2

, Sr2 , Pb

2

+

0

2

-, S2-, OW

A

ll

metals except

In

so lu ble

Ca

2

+, Ba

2

+, Sr2+

CO , PO , SO

A

ll me

ta

lli

c sa

lt

s e

xc

ept Insol

ub

le

BO j- , F , SiO j-

Na

+, K+, NH/

Metal Use

Tab le I - App

li

cation of heavy metals and their t

ox

ic effects

Effect

Hg

Coal. vinyl chl orides. elect

ri

cal

ba ll

e

ri

es

Pb

A I

Cd

Cr

Zn

Co

Se

Ni

Bc

Pl as

tic, pai

nt

, pipe,

ba ll

eries, gasolin e, auto exhaust

Pesticides, coa l. deter

ge nt

s

Ferti I ze r, pl astic, pigme

nt

Tanning,

pa

i

nt s,

pigme

nl

, fun gicide

Fert

il

i

ze

r

Vilamin B- J2

Coal, sulf

ur

Electro plating

Coal, rocket fuel

Neurotoxic

Live

rcirrohosis, me

ni al

disturbance, cancer, ulcer

an

d hypoke

rot os

is

Ki

dn

ey damage, i

nju ry

in cns and me

nt

al re

ta

rda

ti

on

Nephritic, cancer and ulceration

Vomitin g, renal damage and cra

mp

s

Dia

rrh

oea. low blood pressure a

nd

pa ralysis

Damage of li ver, kidney, spl een and nervo usness

Teratogeni c, carc

in

ogenic, genotoxic and mu tagen

Carcinogen, ac ute and chronic poison


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ENDRAN e al.: MICROBES IN HEAVY METAL REMEDIATION

937

bind to many ce llular

li

g

and

s and di splace nati ve

essential metals fro m the ir no

rm

a l binding sites,

whi ch are tox ic

7

. Eubac te

ri

a and

ar

c

hae

a are abl e to

ox idize Mn (II), Fe (II), Co (Il l ), AsO ; , Sea or redu

ce

Mn (IV), Fe (Il l) Co (II ) AsO , SeO on a large

sca

le and

co

nserve ener

gy

in th

ese

r

eac

ti

ons '7. So me

microb es reduce ions such as Hg2+ or Ag+ to HgO and

AgO respec

ti

ve ly,

but

do not co n

se

rve ener

gy

fro m

these r

eac

ti on

3

. Prokaryo tes methylate meta l and

meta

ll

oid compounds produ c ing co rr

es

ponding

volatile metal d e

ri va ti ves

4

. Th e bac teri a l ox idation of

AsO ; to AsO - by a stra

in

o f

Alcaligenes faeca lis

and reduc

ti

on of

CrO

;- to Cr (

OH

h by

Pseudomonas

j luo rescens LB 300 or Enterobacter doc e are examples

of redox reac tions invo lving enzy ma ti c microbial

detoxifica tion of harmful meta ls o r meta

ll

oids

5

.

Microbes secrete in or

ga

ni c metabo

li

c products such

as sulfide,

ca

rbonate o r ph

os

ph ate ions in the ir

respiratory metabo

li

sm and with them prec ipitate tox ic

metal ions as a form of non-e nzy matic detoxifi

ca

tion

6

.

In nature mi crob

es imm

obili

ze

metals throu gh

ce

llul ar

sequestration and acc umulation, or through extrace lliular

. 7

prec lplta

tJOn

- .

Conventional method

of

heavy metal removal from

soil and water

Various me thods are ava il able fo r the remova l and

manage ment of h

eavy

meta ls, which in vo lve tec hni ca l

inputs. Current methods u

se

d for tr

ea

tin g

so il

s

co ntamin ated with tox ic metal are 1) Land filling: the

excava

tion, transport and depos ition o f co ntamin ated

so il in

a permitted hazardous was te land ,

2

Fixa

ti

on:

the che

mi

ca l pr

ocess

ing of so

il

to immobili ze the

metals, usua lly fo llowed by trea tm ent o f the

so

il

surface to e liminate penetration by water and 3

Leachin

g:

us ing ac id

so

lutions as proprietary l

eac

hin g

agent to desorb and leach meta ls fro m

so il

fo

ll

owed

by the return of cl ea n

so

il res idue to site

9

.

Approaches to redu ce heavy metal

co

ntamin ation

in

water include:

1)

precipita tion or fl occul a tion,

foll owed by se

dim

e nta tion and di spo sa l of the

resulting sludge, 2 ion exchange, 3 rever

se

os mos is,

4

microfiltra

ti

on,

5

e l

ec

trodi alys is and

6

eva

poration . Apart from these, other methods like

foa m flo atation, liquid me

mbr

ane te

chniqu

e,

so

lvent

ex traction and crys talliza tio n ca n also be empl oyed .

Bioremediation techniques

Th

e goal

of

microbi al remedi a

ti

on

of

h

ea

vy meta l

contaminated so ils and

sedim

ents are to

imm

obili ze

the metal in situ to redu ce metal bioava

il

ability and

mobility or to remove the metal from the

so

il.

P

az

irandeh et al.

28

described the metabolic path

ways

r

es

ulting in

bioprece

pit ation o f h

ea

vy metals or the ir

biotransformation. Low cos t and higher efficiency at

low metal co nce ntra tions make biotechno log ica l

process ve ry attrac ti ve in compari

so

n to ph ysico

chemica l methods for h

eavy

meta l remova l

9

. Metal

remediation strat

eg

i

es

using microorga nisms ca n

minimi

ze

the bi

oava il

ability and bio tox ic ity

of

h

eavy

metals

3o

.

. Bios timulation, stimulation of viable nati ve

microbi al popul atio n, bi

oa

ugum enta tion, artif ic ial

introducti on o f viable popul a tio n, bioaccumul a tion

use o f live ce

ll

s, and bi osorpti on, use of

dead

microbi al bio mass, are the

cos

t-ef fec ti ve inn

ova ti

ve

bioremediati on t

ec

hno l

og

i

es

. Biolog ica l approac h for

metal detox ifi

ca ti

on affords the potenti al for se lective

removal of tox ic meta ls and operatio n fl ex ibility and

easy

adaptability for

in situ

a

nd

ex situ

appli

ca

ti

on in

a ran

ge

of bio r

eac

tor confi gurati ons

3o

. In the past few

decades, new metal t rea tment and recovery t

ec

hniq ues

based on biosorptio n h

ave

bee n explored us in g both

dead and living microbi al

bi

omass with remarkable

e ffi c iency. Prokar

yo

tic and euk ar

yo ti

c mi

cro

bes are

capable of accumul atin g metals by binding them as

ca tions to the ce

ll

s

urf

ace in a pass i

ve

process

.

Microbes for metal remediation

Th

e mec hani sms by whi ch m

eta

l ions bind to the

ce ll s

ur

face include e lec tros tati c interac tio ns, Van

de

r

W

aa

ls forces, cova lent bonding, redox interactions,

and ex trace llul ar

pr

ec ipita

ti

on, or c

ombin

a

ti

on o f

these processes

3

.The nega tiv e ly charged groups

(carbox y l, hy

drox

yl, and phos phoryl) o f the bacte ri al

ce

ll

wa ll ad

so

rb metal ca tio ns, which are then retained

by mineral nucl

ea

tio n

33

. Bi

oso

rpti on studi es o f U, Zn,

Pb ,

Cd

, Ni ,

Cu

, Hg ,

Th

,

Zn

,

Cs,

Au, Ag,

Sn

and Mn,

showed that the ex tent of so rptio n va ri es markedly

with the metal as we ll as with the microorga ni

sms

(Tabl e

3).

Surf

actants such as rha

mnolipid

produced by

P aeruginosa

show spec ific ity for certain meta ls uch

as Cd and Pb . Hi gher molec

ul

ar weight (- 10

6

)

bi

oe

mul sifiers such emul sa n,

ca

n also aid in me tal

re

mov

al

35

. Studies of Sand

et a f 36

revea l

ed

that

Thiob cilUu s f erroxidans

and

Leptospirillum

fe rroxidans are ca pable of ox idiz in g iron and sulfur.

Joe rge r et al 37 reported that metal acc umulat ing

b

ac

te

rium Pseudomonas stutzeri

AG

259

is

ca

pab le

of

produ cing s il ve r b

ase

d single crys tals, whi ch ca n

reduce the toxicity of metals.


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INDI AN

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EXP BIOl ,

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MB ER 2003

Mechanisms of metal tolerance

Orga ni sms respond to heavy metal stress using

di fferent defense sys tems (F ig. 1) such as exclusion,

co

mpartm enta

li

za

ti

on,

fo

rmation

of co mple

xes and

sy nthes is of binding proteins like meta llothioneins

(

MT

s) and phytochelatins (PCs). Ocha

ri

38

has di vided

ge

neral tox ic ity mecha

ni

sm for metal ions into

three

categories, 1 bloc kin g the essentia l biolog ica l

funct ional groups of biomolecul es

es

pec ia

ll

y

pr

oteins

and enzy mes, 2. displ ac ing the

esse

nti al metal ion in

biomolec ul es and 3. modi fying the ac

ti

ve

conforma tion of bi

om o

lec ul es res ul tin g the loss of

Table 3 - Example of microorgani sms that take

up

heavy metals

Mi croorg

an

isms

Zooglea spp .

Citrobacter

spp

acillus spp

Chlorella vulga ris

Rhizopus arrh i

l s

sp rg

illus

nig

r

Elements

Co

Ni

Cd

U

Cu

Zn

Au

P

Ag

Hg

Th

Pr

otein

denaturation

(Hg,

Pb & Cd .

Up

take

(

dry wt)

25

13

1

70

900

15

14

10

10

54

58

19

spec ific ac

tI

vIty. Mi

croorga

ni sms

ca

n affect h

eavy

metal

co

ncentra

ti

ons in the environment bec ause they

ex hibit a stro ng ability for metal rem

ova

l from

so lution ; thi s ca n be achi eved thro ugh either

enzymati

c or non-enzyma

ti

c mecha

nj

sms

39

.

Avoidance,

res triction of metal entry into the

ce

l

l,

eith

er

by

re

du

ce

d uptake/ac

ti

ve efflu x or by the fo rma

ti

on

of

co mpl exes outside the ce

ll

and sequ estra tion,

reduc

ti

on

of

fr

ee

ions in the

cy

t

oso

l e ither by

sy nthes is

of liga

nds to achieve intrace llu lar chela

ti

on

or by co mpatmenta liza tion are the two majo r

strateg ies of orga ni sms to

pr

otec t themselves against

heavy metal tox icit o .

Th

e ge neral mec hani sms of

metal toleran

ce

in microbes are sh

ow

n in

Ta

ble 4.

Metallothioneine

Meta

ll

othioneines, di

scove

red

abo

ut

45 yea

rs ago,

pl

aya ce

ntra l ro le in heavy metal metabo li

sm

and in

the man

age

ment

of

va

ri

ous forms

of

stres

s.

Meta

ll

othioneines (

MT

s) are low molecul ar we ight

(6 -7 kDa), cystine- rich prote in s, di vided into thr

ee

di ffe rent cl asses on the bas is of their cystine co ntent

and t u ~ t u Th

e C

ys-C

ys,

Cys

-X-

Cys

and Cys-X

X

-C

ys, mo ti fs (

in

which X denotes any amin o ac id)

are charac t

er

istic and invari ant for meta

ll

othi one ine

41

.

Studi es of Lichtl en and Schaf fner

42

show metal

Inhibition of

cell division

(Pb. Cd. Hg & Ni)

/

MI

C

ROB

ES

uption

ell membr

a ne disr

(Hg, Pb. Z

n

N

i.

Cu Cd)

Inhibiti

(Hg.

on

of

enzyme

acti

vity ]

Pb. Zn, Ni. Cu Cd)

Tran

sc

ription inhibition

(H

g

D

N

.

RNA

.

rotein

sy

nt h

es

is

t

t

DNA

dama

ge

Translation

inhibition

(H

g.

Pb, Cd As) (H

g,

Pb Cd)

Fig. I - Heavy metal toxicity mechani sms to microbes


5/10

- 1

RAJ

END

RAN

l

at :

MICROB

ES IN

HEAVY

METAL

REMEDIATION

939

Table 4-Mechanism of metal tolerance in microorganisms

Metal

AsO; AsO/ Sb

J

+

Cd

2

+-

Zn

2

+

Hg2

Co

2

+& Ni2+

Cr0

42.

Cd

2+ Cd2+ Zn2+

r

2

.

Cu

2

+

Toler

an

ce

mechanism

Anion efflux ATPase)

Efflux

AT

P ase)

R

educ ti

on

Efflux

Decreased

uptake

Cat io n effl ux

Decreased uptake

DNA

damage.

reg

ul

atory transcriptional factor-l (MTF-l ) was

essenti al for basal a

nd

heavy metal induced

tran

sc

ription

of

th e stress- responsi

ve

metallothi onein

I and metallothi onein-II. Metallothionein like protein s

have been isolated from th e Cyanobacterium,

Syneococcus spp as well as Escherichia coli and

Pseudomonas putida

4

]

Pl

asmid-encoded energy

dependent metal efflux systems in vo lvin g ATPases

and chemiosmotic ion/ proton pumps are assoc iated to

Ar, Cr and Cd resisstance in Staphylococcus au reus,

Bacillus sublilis Listeria spp, E.co li A.eutrophus,

P.pulida,

some Cyanobacteria, fungi a

nd

algae

7

.

E.coli u

se

a two-component membrane bound ATPase

compl ex, ArsA and ArsB respons

ibl

e for th e act

iv

e

efflux of arsenite form

th

e cell.

In

contrast Gram

positive bacteri a lack th e ArsA protei n

44

.

Genetics of metal resistance

Recent environment pollution with anthropogenic

sources of metals has in creased the need for re search

concerning microbial metal resistance as well as

reme

di

a

ti

on. Large plasm

d

s (165-250 kb), encoded

spec ific metal conferring resistance to a va riety of

metals including

Ag+,

As0

2

, AsO/ , Cd

2

+, C0

2

+,

CrO/

Cu

2

+, Hg

2+,

Ni

2

+, Pb

2

+,

Sb

3

+,

Te0

2

.]

, TI

+, a

nd

Zn

2

+45. Metal resistance systems have been encoded

by chromosomal genes in some organisms like,

Bacillus.s

pp (Hg- resistance) a

nd E.coli.

(A r-efflu

x).

Roane and Pepper

7

catego ri 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 va tion and 3)

general and are ac

ti

va ted by metal stress.

The pl asm id -bo rn e cadA gene encodes a cadmium

spec

ifi

c ATPase

in

severa l bac terial gener

a,

including

Staphylococcus, Pseudomonas, Bacillus

a

nd

E.

coli.

Schmid and Schegel

46

reported

th

e czc and nee

operons are responsible for cadmium re sistance in

Alcaligenes eutrophus CH34 (now renamed as

Ralstonia eUlropha

a

nd

in

A. xy losoxidans,

respectively. The genetic determina

nt

s for heavy

metal re sistance in A.eutrophus CH34 are present in

two pl as mids, pMOL 28 and pMOL 30. Nickel

resistance is encoded by pMOL 28 (163 kb) which

occur by an efflux pathw

ay

via cation proton-

anti porter chemiosmotic system

47

. This removes the

toxic metal , which is accumulated by th e uptake

of

essential divalent cations (e.g. Mg2+,

Mn

2

+ . Taghavi

et al.

48

showed th at nickel resistance is inducible and

is due to an energy-dependent effl

ux

system dri ven by

chemiosmo

ti

c proton-antiporter system.

Genetic engineering for metal. emediation

Genetic engi nee rin g allows introduction of desired

traits into ce

ll

s to metal clean up techniques, and th is

appro

ac

h h

as

already been used to construct ce

ll

s for

the

bi

oreme

di at

ion of mercur/

9

.

Kri shnasamy and

Wilson

o

constructed an E.coli strai n

th

at accumul ated

Ni

2+ by introducing the ni xA gene (cod in g for a

ni cke l transport system) fro m Helicobacter pylori into

E.

coli

1M

109 that ex pressed a glutat

hi

one S-trans

ferase pea metallothioneine fusion prote

in

. The

recomb in ant E.coli strain accumulated four times

more ni cke l

th

an the wild type. Bang et at.

5

eng in eered E.coli to produce sulphide by heterologous

expression of th e thiosulphate reductase gene from

Salmonella entenltca,

which enhanced prote in

synthesis leading

to

th e precipita

ti

on

of

cadmium

sulphide. Metal resitant R. eUlropha isolate,

eng in eered to produce meta

ll

othi onein accumul ated

more Cd

2

+

th

an

it

s wild type co unter pa

rt

, and offered

tobacco plants some pro tec tion form Cd

2

+ when

in oc

ul

ated into contamin ated so il

52.

Studies of

Pazirand eh

28

showed periplasmic ex pression

of

a

Neurospora crassa

metalloth onein in E.

coli

generated

ce lls

th

at were sup erior to bacteria with cytoplasmic

meta llothi one in loca ti on in term s of metal ion

adsorption. Kotriba el al.

53

engineered metal binding

peptides that contain either hi stidines (GHHPHG)2

(HP) or cystines (GCGCPCGCG) (CP) into LamB

and expressed on th e surface of E.coli. Surface

display of CP and HP in creased the bioaccumulation

fo ur and two fo ld , respectively.

54 d b'

Samuelson el al. reporte recom 1n ant

Staphylococcus xylosus

and S.

carnosus,

h

ad

gained

Ni

2

+ a

nd

Cd

2

+ binding capacity a

nd

suggested that

th ey could be used in bioremediation of heavy metals.

They eva

lu

ated surface

di

s

pl

ay systems for expression

of tw o different polyhistidyl peptides, Hi s

r

Glu-His

3

a

nd

Hi s

6

, which had good metal binding acti vity. The

ni cke l resistant bacteria from anthropogenica lly

polluted biotypes and naturally ni ckel-ri ch so ils have

been hybridi zed with various probes carrying cnr


6/10

94

0

IN

DI AN J

EXP

BI

OL, SEPTEM

BE

R 2003

from

pl

as

mid

pMOL28

A. eutrophus

C H34),

nee

from

pTOM

8

A. xylosoxidans

3 1A)

nre

fro m

pTOM8 (A. xylosoxidans

3 1

A)

and

nre

fro m

Klebsiella oxytoea

1

5788. With

res p

ec

t to the ir

h

ybr

idiza

ti

on signals, the nicke l r

es

istant deter min ants

of the stra in s co ul d be assigned to nr nee ty pe,

enrlncc nre

type

Kl ebsiella oxytoca

t

ype

and oth

ers

show in g no hybr idiza

ti

o n

46

. Bac teri al co mmunities in

so il amended fo r many years w ith sewage slud ge that

con

ta in ed h

eavy me

tals we re

assesse

d us in g

mol

ec

ul ar t

oo

ls such

as

, rRNA ana lys is, F ISH

Fluoresence in situ hybridiza

ti

o n), clonin g and

seque nc in g

55

.

iosorption

Biosorpti on, uti

li

za

ti

on of in ex pensive dead or

li ve

mi cro

bi

al biomass to

seq

ues te r metals from

indu

stria l

effl uents has gained impo rtance in recent years due to

their

goo

d pe r

forma

nce, l

ow cos

t, sp

ec

ific ity,

minimu m s ludge genera ti on and amenabili ty fo r

repeated use

34

.

56

.

Several ac

ti

ve

group

s of ce

ll

MICRO E

Pass ive mechanism

Ex

tra ce

ll

ular complexa tion

Ci lrobaCler spp)

Extra cellular po lymeric substance

(S.aure lls)

Po

lysaccharides Z rallligera)

Prot eins A. bisporous)

Nucleic acid

co nstituents like aceda

mid

o group of chitin , stru

ct

ural

pol

ysacc

haride of fung i, a

min

e amin o and

peptid

og

l

ycosid

es),

su

lfhydral and ca rboxy l

gro

ups in

pro

tein ,

phos ph

odi

ester

te icho ic ac id), phos phate,

hydroxy l in polysaccharid es, partic ipate in

biosorptio n

57

. The genera l microbial

med

iated h

eavy

meta l

so

rption m

ec

hani sms a re in the Fig. 2.

Hernandez et ai. 2 iso lated th ree spec ies of bac teri a

be longing to fa mil y Enterob

ac

ter iaceae, whi ch we re

ca pable of

accu

mul atin g nicke l and va nad iu m.

Bi oso

rpti on for metal re mova l has

bee

n stud i

ed

ex tensi ve ly us in g va ri ous spec ies of li ve and

inac

ti

va ted biomass of bacteri a, algae, fung i or

yeas

t1 57 T he tec

hn

ology invo lving su

rface

complexati o n, io n excha nge and mic ropreceip ita

ti

on

is a

po

tenti al alte rna ti ve to current metal tr

ea

ting

processes that ex tend the dura

ti

on of expos ure

of

the

clea

n up

crews

58

. Research in the

area

of h

eavy me

tal

rem

ova

l fro m

was

t

ew a

ter and

sed

iments has f

oc

used

on the developm e

nt

of microbial materi als with

increased

af

finity, ca pac ity and se l

ec

ti vity for t

arge

t

Rhamnolipids

(P.ae ru

ginosa)

Intra ce llular accumulation

(Pseudomollas

spp )

Metal lo

thi

one in

(Synechococcu .I spp)

Precipita tion

CilrobaCler spp)

Efflux pump

(A.e

lllrophu

s)

Acti ve mechanism

Ion exchange reactio n

(S.pia lellSis)

Complexatio n with oxygen and

nitrogen ligands (P. p

Ul

ida)

Destruction of comp lexing

ligands Aci nclOiJacler spp )

Vo latili za tion

Fungal spp)

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

erance


7/10

RAJENDRAN

el al : MI

C

ROB

ES IN HEAVY METAL REMEDI ATl

ON

94 1

metals

,54. Funga l bio mass is finding increas ing

appli ca

ti

on in current biotechnolog ica l procedures for

the cleanup of ni ckel-p olluted effluents and fo r the

r

ecove

ry of metal ions

I.

Raje

ndr

an

et a l 59

reported

A.niger

mut

ant M3

ca

pable of absorbin g 50% more

ni

ckel at 1.7

m co

n

ce

ntration than its parent strain .

Th

e waste fu

nga

l biomass from

indu

strial ferm enters

may

pr

ove to be

cos

t

ef

fec ti ve metal

bi

osorpti ve

age

nts on a lar

ge sca

le

6o

. Fo u

ver

t and R OUX

61

studied

the use of myce li a o f Mucor nicheri,

s

pergillus nige r

and Pencillium chrysogenum fro m fermenters in the

rem

ova

l of nickel, z in c, cadmium a

nd

lead by bio

adsorption. Gadd

31

reported chemica l modifica

ti

on of

bi omass mi ght crea te deri va ti ves with a ltered metal

binding abilities a

nd

affinities. Pse

ud

omonads have

been sh

ow

n to be

ef

ficient in h

ea

vy metal

bi oaccumula tion fro m polluted effl uents in the

imm obili zed state

62

.

63

.

Immobilization for metal remediation

Bi

oso

rpti on t

ec hniqu

e for metal remova l and

accumulati on ca n be even w ith a low deg r

ee

of

und erstanding of the metal

bindin

g m

ec

ha

ni

sms.

H

oweve

r, a be tter

und

erstanding

of

the tec

hniqu

e will

be use ful for e ffect ive and optimized applica

ti

on

of

the method

64

. Fr

ee

ce lls ca n prov ide

va

lu able

in

forma

ti

on in laborato ry ex perim enta

ti

o n but have

li mi tations in industria l appli

ca

tions

65

.

The use of

free ly suspended microbial bio mass suffe rs with

d isadvantages li ke sma ll partic le s ize , low mechani ca l

strength and di fficult y in seques terin g biomass and

effluent.

Th

e

imm

obilized bi

oa

d

so

rbent bi omass

pac ked in sorpti on co

lumn

s, which are

ef

fec ti ve fo r

co

ntinuous rem

ova

l of h

eavy

metals ca n operate o n

cycles co nsistin g

of

loading, regenera

ti

on and

rinsing

66

. Ibanez and U metsu

67

h

ave

demonstrat

ed

the

ability of

pr

otonated alg inate

bea

ds in the rem

ova

l

of

chromium , co

pp

er, zinc, nickel and cobalt ions

from

dilut

e aqu

eo

us so lutions. Bio mass

imm

obili zed in a

range

of

in ert mate ri als like, s il ica, polyacry lamide,

po lymethane and polys ul fone h

as

bee n used in a

va riety of bior

eac

to r

co

nfig

ur

a ti ons, inc luding

rotating bi olog ica l co ntr ac tors, f ixed reac tors, trickl e

filters, flu idi zed beds and air

li

ft bio r

eac

to rs

65

.

Karna

et aL

68

immobili zed

Phormidium va lde rianum BD

U

3050 ] in pol

yv

in yl foa m and u

se

d for the remova l of

C

?+ C 2+ C ?+ d N ?+ 63

a- ,

0 ,

u- a n

1- .

As htana et a l reported

that

Ca

-alg in ate

imm

ob iliz in g agent is ve ry

effec

ti ve

in

ni

cke l bi

oso

rpti on and u

se

d in o r

ga ni

c sa lts like

NaC I and Ca

N03)2 fo r d

eso

rpti on

of

ni cke l fro m

immobili zed mi cr

ob

ia l bi o mass. HC I and EDT A are

the mos t

ef f

ic ient desorpti on agents for ni ckel

rem

ova

I

62

,68.

Plant microbe interaction

n

metal remediation

H

eavy

metal tox ic ity to plants ca n be redu

ced

by

the use of pl ant growth promotin g bac te ri a, free -li ving

so

il bac teria that exert

so

me beneficial

ef

f

ec

ts on

plant dev elopment

wh

en they are appli ed to

seeds

or

inco rporated to

see

ds

70

.

Plants

ca

n

acce

lerate

bioremedia

ti

on in surface so ils by the ir abil ity to

stimulate

so

il microo rganisms thro ugh the re lease of

nutri ents fro m the

so

il a

nd

transpo rt of oxygen to the

rhi zos phere

Plant growth pro motin g bac te ri a that

co ntain ACC dea min

ase

may

ac

t to in sure that the

eth y lene leve l does

not

impa ir roo t gro wth and

facilitate the form ation of large r roots, which enha nce

seedling surviva l

71

Burd

et a l

72

reported that a metal

r

es

istant

so

il b

ac

terium Klu

yvera

ascorbata S

UD

165

pr

o moted the g

rowth

of

ca

nola

Brassica campestris)

in the presence of high co nce ntra

ti

o ns of nickel and

the increase in gro

wth

rate is du e to the abi lity of the

b

ac

te

rium

to lower the leve l of eth y lene stress in the

see

dlin

gs

. Th

ey

further i

so

lated s

ider

ophore ove r

produ c in g

mut

ants, K

ascorbata

165/26 whi

ch

was

able to prov ide suff ic ient ions for the grow th

of

Indian

mu stard

Br

assica j uncea) and tomato Lycopersicon

esculentum) in the presence of high co n

ce

ntrati ons of

Ni, Pb , and Zn . Studies of

Bu

rd et aL 73 sugges t

ed

the

bes t method to prevent plants fro m becomin g

chl

oro

tic in the

prese

nce

of

high levels of heavy

metals was to provide them with a siderophore

pro

du

cing b

ac

te

rium

,

Kluyvera ascor

ba

ta

SUD 165.

Mycorrhi za l fung i also red uce metal tox ic ity to the ir

ho

st plant by

bind

ng heavy meta ls to their

ce ll wa ll

or sur rounding po lysaccharides. Binding sites within

ce ll s of

ec

tomycorrhi za l fung i are meta llot hi onein

like proteins, which h

ave

high cys tine co ntent and

perhaps polyph

os

phate g ranul

es

Field applications

Th e m

os

t

imp

ortant bio t

ec

hno l

og

i

ca

l ap pli

catio

n of

meta l-microbe interac ti o n is in bio leachin g,

bioremediat io n of po lluted s it

es

and minera li za ti on

of

polluting orgnin c matter

l6

l)

ra

nium

is

ext

rac t

ed

fro m

uranit e ore th rough biol

eac

hin g by indir

ec

t

ac

ti on

with T.Jerroxidans . In thi s

case,

ferric iro n in aci dic

so lution, w

hi

ch the or

ga ni

sm ge nerate when ox id iz in g

pyrites FeS2), ox idi zes in so lu ble UO to so lu ble

UO/+

74

. T he

ex

tr

ac

ti on of meta ls such as Co ,

Mo

, Ni,

Pb , and Z n fro m sulfid ie o res by bi oleaching is

tec hnbica lly feasible . Gen

co

r (So uth Africa) is cun 'ently


8/10

942

INDIAN J EXP BIOL, SEPTEMBER 2003

developing tlie BioNIC process to recover nickel from

low-grade sulfide ores

75

.

Signet Technology Inc. and

Signet engineering Pvt. Ltd . (Western Au stralia) are

cUl ently developing a process to recover coba lt from

py rite ore from the kares cobalt project in Uganda

76

.

Various microbially reducible metals, especially ferric

iron

in

com

pl

exed form

to

keep

it

soluble at circum

neutral pH, can be used as terminal electron acceptors

in in situ

anaerobic bioreme

di

ation

of

s ites polluted

w

ith tox ic organi cs

. It is po ssible to bi oremedi ate in

situ s ites polluted with chromate or dichromate [Cr

(Vl)]

by

stimulating th e reduction of

th

e Cr V I) to Cr

(Ill ) by bacte ri a

25

. Fun gi can conve rt oxidized

selenium

to

volatite-methylated selenides for escape

into th e atmosphere, a removal by volatilization

78

Beseker

79

reported Pe

ni

cillium fu.niculasul1l was ab le

to ex trac t more than 50 Ni and 75 Zn from test

so

lu

tions containing

100

mg

rt of

metal at pH

6.6

and

6.

5

respectively. A potent algal bioso rbent Alga

Sorb

T

.

Y

developed using a fresh water alga Ch Iare lla

v

u.lgar

is

and AMT-BIOCLAIMTM (MRA ) developed

us

in

g

Bac illus bi

omass are used to treat wastewater

and me tal recovery respec ti ve l/

6

.

Future prospects

Microbes can significantly affect

th

e di stribution of

metals in th e environment , since th ey have deve loped

mea ns to use

th

em for their benef

it.

This

cl

ea rl y ho lds

promise for effecti ve, eco nomi cal and eco-friendl y

metal bioremediation technology for indus

tri

al

ex pl oitation and pollution free enviro

nm

e

nt.

A good

a

nd

efficie

nt

metal biosorbent can re

pl

ace the

comm ercial ion exchange res in s that have been used

conventionally for metal removal. However, th e bas ic

knowledge of microbi al metal bi oremediation

mec hanism is

in

evitable for the deve lo

pm

ent

of

commercia

ll

y via

bl

e potent bioso

rb

ent. Although

se

vera l

mi

crobi al metal bioremedi at ion approaches

are establi shed to combat heavy metal polluti on from

anthropoge

ni

c and natural sources, none is ye t in wide

spread use. Innovative, economica

ll

y

fea

s

ibl

e and

novel biomass regenerati on and conversion of th e

recovered metal into usa

bl

e form are th e best options

to attract more usage

of

bi oso rb ents. It is time to

II1ttlate

more comprehensive interdisciplinary

approach between biotechnologists and metallurgists

to bring lab sca le

bi

oreme

di

ation process to land scale

tec hn ology that will be acceptabl e for industr iali sts.

cknowledgement

On e of th e authors CPR) is indebted to UGC, New

Delhi fo r

th

e awa rd of Teacher fe llows hip for doctoral

studies and to the Management

of

Vivekananda

College, Thiruvedagam, Madurai for deputation under

F.I.P. program.

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