Indian Journal of Experimental Biology
Vol. 41 , September 2003, pp. 945-966
Microbial biomass: An economical alternative for removal of heavy metals
from waste water
Rani Gupla* & Harapri ya Moh apatra
Department o f Microbi ology, Uni versit y of Delhi South Campus, Benito Juarez Road, Ncw Delhi 110021. Indi a
Today indi sc riminate and unc ontrolled di schargc of metal contaminated industrial effluents into the environmclll has
bccome an issue of major concern . Heavy metals, being non-biodegradab lc and persistent , beyond a permi ssible
concentration form unspec ifi c compounds inside the cells thereby causing cellular tox icity. Thc onl y alternative to rcmove
them from the wastewater is hy immobi li zing them. The conve ntional meth ods adopted earli er for thi s purpose included
chemical prec ipitation, oxidation, rcducti on, filtrati on, electrochemi cal treatment, eva poration , adsorpti on and ion-exchangc
resins. These methods require hi gh energy inputs es peciall y when it refers to dilute solu tions. Here microbial biomass offcrs
an economical option for rem ov ing heavy mctal s by th e phcnomenon of biosorpti on. Non -li ving or dead biomass sequestcr
metal (s) on thcir ce ll su rface duc to certain reactiv c gro ups available like carboxy l, ami ne. imidazole, phosphate,
sulph ydryl, su lfate and hydroxy l. The process can be madc economical by procurin g spcnt biomass from industry or
naturall y ava il ab le bulk biomass. A batch or a cOlllinuous process o r remo val of heavy metal s dircctly from effluent s can be
developed in a fi xed bcd reac tor using the immobi li zed bi omass. Further biosorpti on potential of th e biomass can be
improved by vari ous ph ys ical and chemica l treatmcnts. Thc avai lab ility of variety of microbi al biomass and th eir metal
binding poteilli al makes it a cconomical and sustainable option for developi ng effluent treatment process fo r remo va l and
recovery of hea vy metal s.
Keywords: Effluent treatment , Heavy metals, Metal binding potential , Metal pollutant , Microbial biomass
A sudden boost in th e indu strial activities has
contributed quantitatively as well as qualitative ly to
the alarming increase in the di sc harge of metal
pollutants into environmental sink , especially the
aq ueo us e nvironment. Di spe rs io n of the metal ion s in
water bodies leads to their bi omag nification through
the food cha in and results in increased toxicity. This
fact renders the removal of heavy metal s from
aqueou s sol ution s indispe nsable. Thus, the boon of
affl ue nce has in turn given ri se to the curse of
effluent. More than fifty percent of the heavy metal
pollution has been accounted to the anthropogenic
act iviti es. The tragic episodes of ' Itai- Itai ' and
' Mina mata ' brought into focus the global concern
regarding th e impact of environmenta l pollution o n
human hea lth . Since then severa l centers all over the
world have been e ngaged in th e deve lopme nt of
processes for re moval of heavy metals t •4 .
Th e conventional processes used for effluent
treatment are precipitation as hydroxides/sulphides,
ox idation/reduction , and Jon exchange. Th ese
* Author for correspondence:
Phone: +9 1-01 1-61 11 933
Fax: +9 1-0 I 1-6885270
E-mai l: microzyme@ 123 india.com
processes are expe nsive especially when the
contaminant metals are dissolved in large volumes
l
and appear in the concentration range 10-100 ppm .
Moreover these are not ecofriendly in nature and
result in the production of large amount of sludge. As
a result an aquatic problem is transformed into a olid
waste di sposal problem. Therefore, a mo ngst the
chemical adsorbent o nly ion exchange resi ns were
considered as th e option for remediation with least
ecological probl e ms. However, chemical res in s are
expensive and the increasing demand for eco-fri e ndly
technologies has led to the search of low cost
alternatives that could be considered as single use
materials. In thi s li ght, biological material s have
6
e merged as an ecofriendly and economic option . For
a long time acti vated carbon and peat occupied the
place of promine nce among biosorbent' s but since
they were geograp hica lly restricted in distribution
microbial biomass became the unanimou s choice.
Microbial biomass can bind heavy metal s e ither
actively or pass ively o r by a combination of both
7
processes . The passive phenomenon of ' bioso rpti on'
has several advantages over the active phe no menon of
'b ioaccumul ation' . Growth
physiology of an
organism varies drastica lly with variation s in the
effluent compositi o ns making it difficult to ex press
946
INDIAN J EXP BIOL, SEPTEMBER 2003
mathematicalll -IL.. On the other hand , biosorption
involves use of non-growing biomass/dead biomass
for sequestration of the metals, thus the process is
independent of metabolic activity. Another major
disadvantage of bioaccumulation is recovery of the
accumulated metal by destructive means whereas in
biosorption desorption is accomplished by simple
ph ys ical method s without damaging the biosorbent's
structural integriti I. Moreover, biosorption has an
edge over bioaccumulation with easy and cost
effective procurement of the biomass either as a byproduct of a large scale fermentation process or bulk
procure ment from natural water bodies I. Last but not
the least being a surface phenomenon, most of the
biosorption is generally completed within few min of
contact with the biomass.
Several work ers l.3-5.12- 15 have presented exhaustive
review s on the biosorption.
Microbial biomass: taxonomic considerations
A wide variety of biological materials have been
. d for t helr
' meta I b'IOsorptlon
.
.. I 16.17.
exp IOIte
capacities'
One can begin the count down from the classical use
of activated carbon to peat and end up with the
. b'Ia I b'losorbent matena
. Is 181
WI'desprea d repOits 0 f mICro
. 9.
Fungi, yeast and bacteria as by-products of industrial
fermentation processes while macro algae due to bulk
availability of their biomasses from natural water
bodies have attracted most of the attention as is
evident from the literature 2o-25 .
Fungi and yeasts belonging to the genera Rhizopus,
Aspergillus, Penicillium and Saccharomyces have
26 3o
shown excellent metal biosorption capacities - .
3
Muraleedharan et al. 0have screened several tropical
wood rotting mu shrooms for Cu biosorption.
Ganodenna lucidum have shown good sorption
31
potential for Cu and rare earth metals . Karavaiko
et al. 32 have reported selective extraction of noble
metals from solutions by microfungi, yeasts and
actinomycetes. Uranium biosorption potential has
been reported for polyester entrapped foam beads of
Trichoderma harzianum 33. Among bacteria, Bacillus
subtilis has been identified to have potential for
metal sequestration and has been used in
3 26
the commercial biosorbent preparation . . Sahoo
34
et al. have reported good Cu and Cd removal
potenti al for Bacillus circulans. Besides, there also
exist reports on the biosorption potential of
Pseudomonas sp. , Citrobacter sp., Zoogloea ramigera,
Arthrobacter and Streptomyces spp.
Though available as a cheap industri al by-product,
one major disadvantage of using biomasses from
industrial processes is the presence of impurities due
to the adhering fermentation broth resid ue th at might
interfere with its metal sorption capaciti 5 . Dias
et al. 40 have compared the biosorptive potential of
waste biomasses obtained from four different
di stilleries. They observed marked vari a tion in metal
binding capacIties of these biomasses. These
variations could be attributed to diversity in the
microflora composition and culture conditions in each
41
of these production media. Omar et al. in their
studies on uranium removal by the brewery yeast
Saccharomyces cerevisiae observed hi gher U removal
by unwashed, dried and ground yeast as compared to
its washed and live counterpart. Waste products of
antibiotics industries were able to remove 99 % Zn
and Cd and 95 % Ni from the metal solutions 25 .
Further, in case of bacteria, immobilization of the
biomass prior to its application in continuous
columns/fixed bed reactors is must. Exhaustive lists
of the metal sorption capacities of heterotroph s have
been presented by3.13.42. Mc Lean and Beveridge43have
reviewed the metal binding capacity of bacterial
surfaces.
Besides heterotrophs, photoautotrophs especially
seaweeds have been extensively worked upon . The
brown seaweeds belonging to the gener(} Ascophyllum
and Sargassum form the most exploited organisms for
. h Ig
' h
'
.. ? I 22"
44 45. Th e ot h er
t helr
metaI
sorptIOn
capacltles-'
exploited groups include Fu cus versiculosus, Eisenia,
Laminaria, Spirulina , Porphyra, Cyanidium. Among
the green algae the genus Chlorella has been
identified as a potential candidate for metal
biosorption 46-48 . Gale49 has reported effective removal
of Pb by blooms of Chlo relia , Cladophora ,
and
Rhizoclonium,
Hydrodictyon,
Spirogyra
Oscillatoria. Kuyucak and Voleskyll have evaluated
gold and cobalt removal abilities of green (Cosium
taylori, Halimeda opuntia), brown (Macrocystis
pyrifera, Undaria sp., Laminaria sp. Ascophyllum
nodosum, Sargassum jluitans, S. natal1s) and red
(Chondrus crispus, Palma ria palmate, Porphyra
tenera) marine algal species.
Though the potential of these algal cultures to
remove heavy metals under laboratory conditions
have been well established, the major bottleneck lies
in their commercialization and large-sc ale production .
Recent developments in algal biotechnology have
resulted in the commercial production of certain
strains for their use either as animal feeds, ch emi cals ,
GUPTA & MOHAPATRA : MI C ROBIAL BIOMASS FOR REMOV AL OF H EA VY META LS
biochemicals or fertilizers or as a source of food for
humans so . The commerci a lly g rown gene ra are
limited in number (only 8 gene ra) and include
Spirulina, Chlorella, Scenedesmus, Phaeodactylum ,
Botryococcus, Chlamydomonas, Dunaliella and
Porphyridium . Of th ese, o nly Spirulina and Chlorella
have been produced co mmerc iall y, e xc lusive ly fo r
so
their metal remova l capaciti es . Unlike fung i, yeast
and bacteri a, the co mme rci a li zed a lgal g ro up has the
advantage of be ing a better defin ed materi a l as the
conditi ons in the natural water do no t change as mu ch
as those of the culture medi a.
Abo ve rev iew of the lite rature revea ls that apart
from fe w genera such as Spirulina, Nostoc ,
Cymodocea and Scenedesmu s the re ex ist very fe w
s, ss
repo rts of cyano bacteri a in the area of bi osorpti o n - .
Developing biosorbents for commercial application:
Immobilization and reinforcement
Despite the tre me ndo us pote ntia l of the a lga l cell s
for heavy metal re mo val they still face maj o r
chall enges for be in g expl o ited co mme rc ia ll y .
Commercial biosorbe nts need to fulfill a number o f
criteri a such as: (i) hi g h bi osorption capac ity at
equilibrium i. e. th ey sho uld co ntain as little as
poss ible of inert materi al in the ir binding sites (ii )
fa vorabl e biosorpti o n kineti cs i.e. particl es sho uld be
hydrophilic and po ro us in nature (iii ) maintenance of
smooth fl ow d ynami cs in a reacto r - thi s prevents the
use o f either very small o r strong ly swe llin g particl es
in the co lumn (iv) amenabl e to rege nerati o n - thi s
necess itates desorption by minima l poss ibl e vo lume
of desorbin g agent w ithout damag in g th e bi oso rbent
(v) good mechani cal streng th (v i) te mpe rature
stability and (v ii ) res ista nce to che mi cals s6.
The tendency of the free algal ce ll s to clump
together leads to cl ogg ing of the column. Thi s al so
necess itates excess ive hydrostati c pressure to generate
suitabl e fl ow rates. Mo reover, the frag ile nature of the
free algal cell s re nders them susceptible to
di sintegrati on due to hi gh pressures. All these
limitations make it imperative to immobili ze these into
suitable matrices pri or to the ir use as co mmercial
s7
biosorbents in columns . Not all algae require
immobili zation; macro algae such as Sargassum or
Chara need onl y pro per sizing pri o r to use while all
micro algae require some degree of immobili zati on o r
pre-treatment or hardening. Immobili zatio n o f the no nviable cell s has been carri ed o ut in several matrices
natural as well as sy nthetic . T able 1 li sts the use of
various immobilizing matrices fo r metal bi osorpti on .
947
Among the most commo nl y used matri ces are
polyacrylamide, calcium/sodium a lg inate and silica.
Other matri ces may inc lude sta inl ess steel wire, pan
scourers, cotto n webbin g, alumin a, coal, foa ms o r
36
po lyviny l chl o ride. Philip et al. have repo rted the
use of several commo nl y ava il abl e, inex pensive
matlices for immobili zation of Pseudomonas aeruginosa
ce ll s. These included a lumin a, coco nut she ll , g irid ih
bituminous coal, g lass, cerami c materi a l, refracto ry
bricks, rice hu sks, sili ca gel and sand . S ili ca
immobili zed matrices posses' g reater mechani ca l
strength coupl ed with exce ll ent fl ow c haracte ri stics .
The co mme rc ial alga l biosorbe nt Al gaSORB™
developed by Bio-Recovery Syste ms, Inc., utili zes
7s
sili ca as the immobili zati o n matri x . Hi gher metal
re moval abilities by immo bili zed o rgani sms have
been repo rted by severa l worke rs 70.7 0. Aspe rg illus
niger my celi a immobili zed in po ly ureth ane fo am
sho wed three-fold inc rease in C u remova l as
7o
compared with th at o f free cell s .
.
38 7? 73
.
Res rns such as po ly sul fo ne . -, and epoxy res In
s 7'have also been success full y used fo r e ntrapment and
immobili zation. Polyvinyl alco ho l (PV A) immobi li zed
beads of Phormidium valde rium were abl e to re move
80% and 60% Co pe r g ram in 24 hr60.
Se lecti o n of matri ces sho uld be do ne such th at they
do not hamper o r slo w do wn the so rpti o n capac ity o f
the bi o mass by bl ocking or co nsuming the bi nd ing
39
sites. Park e f al. observed a decrease in bi oso rpti o n
fo ll o wing immobili zati o n of Zoogloea ramigera cell s
in calcium a lgin ate. Park et al.59 have a lso repo rted
lo we r Cd uptake by Ca-a lgin ate e ncapsul ated
Aureobasidium pullulans th an by the free bi osorbent.
Apart fro m immo bili zati o n, re inforceme nt and
cross-linking also pro vides a suitable mea ns of
stabili zing the bi osorbe nt. Cross-linking can be
achieved by formalde hyde, glutera ldehyde and/or
po lyeth ylene imine. Hi g her durability was reported
for cross-linked cell s (tri ethy lene tetra mine and
39
g lutamic di aldehyde 1% w/v) of Zoogloea ramigera .
The autho rs have repo rted the cross-linked capsul es to
maintain the ir mec hani cal stre ngth and adso rpti on/
desorpti o n capac ity e ven after 30 cycles of re peated
use. Ashke nazy et al.77 have repo rted be nzaldehyde in
aceto nitrile to be a good fi xati ve, whi ch reta in ed the
initi a l Pb bi osorpti ve pote nti al of Saccharomyces
cerevisiae . Fixation with be nzaldehyde in aceto nitril e
a lso re ndered the bi o mass res istant to mi crob ial
spoil age fo r up to 1 yea r at 4°C.
Some workers have a lso repo rted the pre-treatment
of the bi osorbent w ith acids, alka li , salt so lut io ns,
INDI AN J EXP BIOL , SEPTEMBER 2003
948
detergents or organi c so lve nts to affect the b iosorptive
potential of the biosorbent 2 1.78.79 . Drying of the
biosorbent materi al has reported to have different effects
on the bi osorptive potenti al as e vident form Tab le 2.
Process parameters affecting biosorption
The bi osorption process being a cell surface
phenomenon is to a large exte nt affected by the
chemi cal compos iti on of the cell surface. Apart from
the vari ati ons in the c he mica l nature of the bi osorbent ,
th e process is also affected by a vari ety of other
physio-c he mi ca l paramete rs such as pH , te mpe rature,
initi al metal concentration , time of contact, co-ions
and bi osorbent concentrati on. A compre hensive detail
of the vari ous parameters affec ting the bi osorptive
capac ity of th e bi osorbents has been listed in Tab le 2.
pH
p H of the e nvironment influences the biosorption
process in several ways. Th e most important effect is
Table I -
the change imparted by it on acti ve b indin g s ites,
which in biosorpti on are usuall y acidic in nature.
Dec rease in pH leads to their proton ation thereby
decreas ing their negative ch arge and consequentl y
the cation binding . On the othe r hand , a n increase
in p H increases the avail abi lity of the negati ve ly
charged free sites for electrostatic attraction of
cati ons, thereby resulting in an inc rease in the cati on
binding capac it/ 5 . Sal' et al.35 ha ve reported
max imum sorpti on of Cu at pH 7 .0 whi le in case of
Ni the tre nd continued till pH 8.0 . V iann a el al.89
have reported low bindin g of Cu , Cd and Zn at ac idi c
pH (2.5and 3. 5) whil e the non-protonated bio mass of
Bacillus lenlus and Aspergillus oryzae showed hi gher
Cd sorption , 80 and 20 mg/g, respecti ve ly. Puranik
a nd Paknikar38 have reported pH op tima of 4.5,
6 .0 a nd 6 .5 for Pb, Cd and Zn uptake, respectively
by Citrobacler strain MCM B-1 8 1. A pH va lue
of 5 .8 was found to be optimum fo r Ag sorption by
I mmob ili zati on matri ces/condit ioning used in metal biosorpti on
Immobil izati on matri x
Organi sm
M etals adsorbed
Lyophi l ized bi omass
Pseudomonas aeruginosa
Asperg illus niger, Penicillium chrsog enum,
Mu cor miehei
Pseudomonas aeruginosa
U
U
37
58
Ni . Cu
35
Zoogloea ramigera
Tremetes versicolor
Cd
Cd
Cd
59
39
23
Phormidium valderium
Sargassllln sp.
Cymodocea nodosa
Sa rgasswn spp .
GloeOlh ece magna
Azalia fi liculoides
Rhizopus a rrhizus
Cd 2+, Co 2+ , Cu2+, Ni2+
Cd, Zn
Cu, Zn
Cd, Cu
Cd, Mn
Ni
Fe(CN ),)
60
61
62
Polyacrylamide
Chlorella regularis
Streptomyces viridochrol1logenes
Pseudomonas EPS 5028
U
66
67
68
Silica
Chlorella vulgaris (Al gaSORB TM )
A u, Cu, Ni, U, Pb, Hg, Cd,
Zn, Ag
107
Polyureth ane foam
Rhizopus oligosporus
Aspergillus niger
Rhi zo pu s a r rh i z u s
Cd
69
Cu
70.7 1
Bio-Fix (consortium)
A I)+, Cd 2+ , Zn2+ and Mn 2+
Phorl1lidiwn lam inosum
Cit robacter strai n M CM B - 181
Mu co r r Ollxi i
Cu 2 + , Fe2+, Ni 2+ and Zn2+
Pb, Cd , Z n
Phormidiwn laminosw l1
Ellferobacler
Cu2+, Fe2+, N i 2+, Zn2+
Ni 2+
Ca lcium/Sod iu m alginate Aureobasidium pullulans
Polyv inyl alcohol
Dry ing
Polysulfone
Epox y res ins
M ag netit e (Fe)0 4)
References
22
63
64
65
3
72
38. 73
72
74
.J
-..-
~
~
-+
Table 2 - Effect of process parameters and biosorptive potential of some microbial biosorbents
Organism
Aspergillus niger
Time
kinetics
pH
Temperature
I hr
5.0
30°C
Biosorption
capacity
Biomass
pretreatments
14-15 mg Cu 2+/g
Immobilization on
polyurethane foam
increased biosorption
Effect of
cations/anions
Eluant
EDTA (I mM)
Reference
70
Cl
C
Chlorella vulgaris
Sargassum fluitans
Zoogloea ramigera
15 min
10-20 min
6.8
3.1 mg Znlg
4.0
0.94 mmol/g
6.5
37°C
Spirogyra sp.
2hr
2.0
18°C
Sargassum sp. 1 to 6
6hr
4.5
22°C
2.21 mg Cdlg (free
cells); 1.78 mg
Cdlg (Ca alginate
immobilized); 1.11
mg Cdlg (bead
entrapped)
Protonated and
crosslinked with
fonnaldehyde
Cymodocea nodosa
Citrobacter strain
MCM B -181
2hr
20 min
5 min
4.5 (Pb)
HCI (1 M)
Rhizopus o/igosporus
20 mi n
5.0
Streptomyces rimosus,
Penicillium
chrsogenum,
Saccharomuces
car/sbergensis,
Saccharomyces
cerevisiae
15 min
6.0 (Cu)
7.0 (Zn)
8.0 (Ni)
0
80
0.9 to 0.66 mmol
Protonation with HCI
Cdlg; 0.93 to
(0.1 M)
~Co
>Sr
CaCI 2 (1%),
Ca(NOJh and
HCI (0.1 M) at
SIL ratio 1.0 gIL
22
EDTA (50 mM)
81
Cll
)::
r
to
(5
s::
»
en
en
62
'Tl
0
;:0
;:0
tTl
s::
0
Several chemical
treatments increased
Cd sorption but not
Zn and Pb. Biomass
could be immobilized
on polysulfone
Pb>
Zn>Cu>Cd>Ni>Co at
pH4.5
Zn>Cu>Cd>Ni>Co at
pH 6.0
EDTA (0.1 M)
HCI (0. 1 M)
38
Penicillium biomass
was acid treated
<
»
r
0
'Tl
:c
tTl
»
<
-<
82
126 mg Pb/g
More than 90%
removal
39
»
-0
»
-l
s::
n
;:0
52.68 mg Culg;
46.56 mg Znlg
30°C
Ro
;:0
14.7 mg Cdlg
23.6 mg Znlg;
43.48 mg Cdlg;
58.78 mg Pb/g
s::
»
Cd >Ni
28°C
21
:c
Cross linking with
1% (w/v) triethylene
tetramine & 1%
gluteric sol ution
increased durability
9.0
4 .5 (Cu)
5.5 (Zn)
»
0
0.89 mmol Culg
Bacillus simplex
~
25
Zn >Cu>Ni>Ca>Na
83
s::tTl
-l
»
r
en
(- Collld)
'-0
.p.
'-0
\0
Ul
0
Table 2 - Conld
Organism
Time
kinetics
Rhodymenia palmale,
Porphyra yezoensis,
Laminaria japonica,
Eisenia bicyC/is,
Macrocystis pyrifera,
Cyanadium caldarium,
Spirulina platensis,
Chlorella pyrenoidosa,
Chlorella vulgaris
Saccha romyces
cerevisiae
5 min
5 min
5 min
60 min
5 min
5 min
5 min
5 min
5 min
15 min
2.0 to 6.0
2.0 to 6.0
6.0
2.0 to 4.0
6.0
2.0 to 6.0
2.0 to 6.0
2.0 to 6.0
4.0-6.0
10 min
30°C
5.0
Biosorption
capacity
Biomass
pretreatments
40.11 mg Aulg by
R.palmate; 83.5 mg
Aulg by
C.caldarium; 70.68
mg Aulg by
S.platensis;
97 .6 mg Aulg by
c.pyrenoidosa
C. vulgaris could be
immobili zed on si lica
20 min
2.0
Rhizapus arrhizus
IS min
6.0 to 7.0
(U233 ,
Pu239),
2.0
(A m24 1,
Cel44,
Pm147,
Eu152154,
Zr95)
Room
temperature
..J...
Effect of
cations/anions
Eluant
Thiourea
(0.1 M) pH 2.0
Reference
20
Z
:;
z
0
Pretreatment with
NaOH & SOS
decreased sorption
104.8 /lmo l Aglg
Cd & methionine
decreased Ag sorption;
Na+ & SO/ ' at a
concentration 1000
fold excess th at of Ag
had slight inhibitory
effect.
190 mg Co/g
84
Cu 2+,
A1 3+,
Fe 3+,
tTl
X
"0
OJ
0
.r
Vl
tTl
~
HCI and
H2 SO. (0.1 N)
541 mg Ulg
(lyophilized cells);
410 mg Ulg (live
cells)
Azalia filiculoides
"1'
Biosorption
by
Chlorella
pyrenoidosa
was highly
temperature
dependent:
increased
with
increase in
temperature
with a
maxima at
60°C.
4°C
7.0
Rhizopus spp. Based
biosorbent PFBI 2
Pseudomonas strain
Temperature
pH
tTl
85
3::
OJ
tTl
;;0
Fe 2+
37
tv
0
0
and Th4+ inhibited U
so~tion . CO/' and
SO ' at 1000 mdldm3
inhibited U sorpti on
w
99.9% removal of
Au
45
Biosorption
capacity me~s ured
in terms of
distribution ratio
U-3071, Pu-9653 ,
Am-8196, Eu-7113,
Pm-9386, Ce-9448,
Zr- 1995.
Biosorbent stabl e at
pH 2.0 and 11 .0
cr, N0 3., SO" , C03.
HN0 3 (2 M)
86,87
and CH 3COO' (O.IM)
decreased Pu sorption
(-Contd)
...
....
..,..
Table 2 - Contd
Organism
Trametes versicolor
Phanerochaete
chrsosporium
Time
kinetics
60 min
2 hr
pH
Temperature
5.0 to 6.0
Biomass
pretreatments
102.3 mg Cdlg (live
entrapped cells);
120.6 mg Cdlg
(dead fungal
mycelia)
Heat treated dead
entrapped cells
showed higher
biosorptive capacity
2.4 rrunol UO/+/g
Saccharomyces
ce revisiae
60 min
Rhizopus nigricans
30 min
2.0
45°C
47 mg Cr/g
Azolla filiculoides
10 min
6.5
18°C
43.4 mg Nilg
Pseudomonas
aeruginosa
(lyophilized)
10 min
7.0 (Cu),
8.0 (Ni)
Temperature
independent
37°C
Effect of
cations/anions
Eluant
Reference
C
HCl (10 mM)
23
>
Ro
3::
0
::c
>
....,
24
>
-l
::0
>
3::
41
Dry cells performed
Ni sorption : Cd most
antagonist; Fe, Cu,
Co and Pb caused
some degree of
inhibition; Cr or Zn
had marginal effect;
Na, K & Ca enhanced
sorption.
Cu sorption: Cd most
antagonist; Inhibited
slightly by Zn, Extent
of inhibition to the
same extent by Fe &
Co; Na, K&Ca
increased sorption.
0
:;
88
Alkali reconditioning
improved adsorption
capacity
n
::0
O:l
better than live cells
265 mg Nilg,
137.6 mg Cu/g
a
:::j
Affinity Cu>Pb>Cd
23.04 mg Cdlg;
69.77 mg Pb/g;
20.23 mg Cu/g
6.0
4.5
Biosorption
capacity
Slight inhibition
observed in presence
of Ca 2+
HCl and
H2S04 (0.2 N)
64
r
O:l
0
3::
>
C/)
C/)
'"r:I
0
Pretreatment with
NaOH, N~OH&
toluene increased
metal sorption. Oven
heating (80°C),
autoclaving, detergent
and acetone treatments
decreased sorption
HCl, H2SO 4 ,
HN0 3 and
nitiloacetic
acid (0.1 N) ;
CaC0 3 (10
mM)
35
::0
::0
tTl
3::
0
<
>
r
0
'"r:I
::c
tTl
>
<
-<
3::
tTl
-l
>
r
C/)
'D
VI
INDIAN J EXP BIOL, SEPTEMBER 2003
952
84
Saccharomyces cerevisiae . Hi ghest specific lead
uptake by Rhizapus oligosporus was obtained at pH
5.082 . Zakharova el a l?5have reported an increase in
th e degree of metal sorption with an increase in the
solution pH for a number of mi crobial biosorbents
(bac teria, fungi, yeast, asco mycetes and algae) tested.
Optimum pH for remov al of U by Saccharomyces
41
cerevisiae was found to be 4.5 . Optimum pH for
biosorption of Cd , Pb and Cu by filamentous fungus
24
Phanerochaele chrysosporium was found to be 6.0 .
37
Sar and D' Souza observed maxim um U uptake by
Pseudomonas aerug inosa at pH 5.0 while the sorpti on
was negligible at lower p H. They attributed thi s firstl y
to the hi gh solubility of uranyl ions and co mpetiti on
by W for U binding sites in th e ac idic co nditi on,
seco ndly increased bindi ng affinity of the biomass for
monovalent uranyl species rU0 20H+, (U0 2)\OH s)]
fo rmed at hi gher pH (4.0-5.0) over th e diva lent
(UO/+) at low pH (2.0) and thirdl y to a dec rease in
di ssolved uranyl ion co ncentrati on at p H above 5.0
due to fo rmati on of so lid scheopite (4UO,.9H20 ).
Maximum biosorpti on of Cd ions by Ca-a lgin ate
immob ili zed Tram etes versicolor occ ulTed between
pH 5.0 and 6.0. Beyond 6.0 there was a drastic
dec rease in sorpti on , whi ch could be attributed to the
format ion of cadmi um hyd rox ide co mplex 23 . These
references exemplify a second way by which th e pH
affects metal sorption. Certain meta ls occ ur in th ei r
free and hydrated form at lower p H and as their
hydrox ides at hi gher pH. This leads to precipitation
and thus red uced avai lab ility of the metal ions. In
such cases it is important to co nsider seve ral species
of one metal ion as an individual sorbate. As sorpti on
increases with dec reasing solubility, hydro lyzed metal
ions are eas il y sorbed than free metal ions. Less
energy is required to re-o ri ent th e hydrated water
mol ec ul es of these less hydroph ilic meta l ions IS.
Still other metal ions such as Au, Cr, Hg, and Ag
have a greater tendency of occ urrin g as negatively
charged complexes e.g. AuCN', AuCI"4as in case of
Au. Such metal ions either show a decrease uptake
with increasing pH or remain inse nsitive to pH
change. Maximum removal of go ld by Azalia
45
jllicilloides was observed at pH 2.0 . The authors
have attributed thi s to the anionic nature of AUCI"4'
Bai and Abraham 88 observed increased sorption of Cr
(V I) by Rhizopus nigricans biomass at pH 2.0. They
attributed this to th e predomi nance of anionic spec ies
of Cr (HC r0"4, Cr20 \, Cr40 20 13 and Cr20 20 10) at thi s
low pH, which co uld eas il y in teract with th e
positi ve ly charged cell wall li gands. Maximum Co
removal ( 190 mg/g) by Rhizapus spp. based
biosorbent PFB I was obtained at p H 7.0 85 . They
however observed an average bi osorption of 85 % in
the pH ran ge 2 to 8.
Time kinetics
The major adv antage of the biosorptive metal
uptake is its fast equil ibrium kinetics. Bioso rpti on
generally co mpl etes within fi rst few min of th e in itial
co ntact of biomass with th e metal bearing soluti on,
Following thi s, equi librium is es tab li shed between the
metal uptake by the biomass and the residu al metal in
the so luti on. Rapidi ty of th e process of biosorption
seems to be consistent with the mechan ism of passive
adso rption to the cell s rat her than being a
metabolica ll y acti ve process 4 .90 ,
Blanco el al. 72 have reported a relative ly hi gher
time of co ntact (60 min ) wi th 38-65 % recovery of
heavy metals, viz. Cu, Fe, Ni and Zn by polysulfone
and epoxy bead immobili zed Phonnidiul11 laminoslllll.
Sal' and D'Souza 37 have repo rted 90% of U load ing to
occ ur within 10 min of contact with li ve
Pseudomonas cells. Eq uili brium was reached with in
60 min of contact in case of li ve cell s whil e
lyop hili zed biomass showed much slower rate and
reac hed satu rat ion onl y after 120 min. Majority of Cd
so rpti on by free Aureobasidium pullulans biosorbent
was establi shed within 10 min of expos ure59 Gold
removal by Azalla jlliculoides was rapid with nearly
80% of the metal being removed within 20 min of
expos ure at p H 2.0 45 , Compared to th e above system
U sorpti on by Pseudomonas aerugin osa takes a mu ch
longer time with 50% sorpti on tak ing place after I hr
of ex posure36 and equilibrium being reac hed after
2 hr. Pb biosorption by Rhizapus oligosporus was
very rapid during th e initi al stages of sorpti on process
(0-20 min ) but reac hed equi li brium onl y after 14 hr of
ex pos ure 82 . Time kinetic studi es of Citobacter starin
MCM B-1 81, revealed the metal upta ke to be rap id
with 85 % Pb, 70% Cd and Zn being adsorbed in the
first 5 min 38 . Cadmium biosorption by Zoog loea
ramigera reac hed eq uilibrium fo llow ing 10-20 min of
expos ure39 . For all the mi cromycetes rested for their
Ag so rpti on, hi ghest rate of the process was observed
during the first min of incubati on reac hing th e
g8
eq uilibrium within 20 min 91 . Bai and Ab raham
observed rap id adso rpti on of Cr ions durin g initi al
30 min of so rbent contac t in case of Rhizopus
41
nigricans biomass. Omar et al. in th eir studi es on U
removal by Saccharomyces ce revisia e observed
max imum removal to occ ur during first 60- 100 min of
GUPTA & MOHAPATRA : MICROBIAL BIOMASS FOR REMOV AL OF HEAVY METALS
24
exposure. In contrary to the above reports, Say et al.
reported more th an 60% adsorpti on of Cd, Pb and Cu
followin g an ex posure for 2 hr 111 case of
Phaenerochaete chrysosporium. Hi gh initi al Cd
biosorption rates were observed for Ca-alginate
immobili zed Trametes versicolor at the beginning,
whi ch approac hed equilibrium within 60 min of
ex posure23 .
External metal concentration
Initi al metal concentrati on plays an important ro le
in determining th e bi osorpti ve capacity of a
biosorbent. Generall y, it has been observed th at an
increase in the initi al metal co ncentrati on res ults in an
increase in the metal sorpti on capac ity of the
biosorbent, whi ch culmin ates in a pl ateau at very hi gh
metal co ncentrati on. The metal so rpti on capac ity of
an organi sm touches its pea k at th ese hi gh metal
concentrati ons. At this point th e sorpti on of the metal
is limited by th e ava il ability of the number of binding
sites 111 the bi omass . Thu s, at low metal
concentrati ons (as enco untered in efflu ent sampl es)
the biosorption ca pac ity of the bi osorbent is not full y
utili zed l5.
An increase in the specific metal uptake with
increase in initial metal co nce ntrati on was observed in
case of Citrobacter strain MCM B_1 8 138 . More th an
90% of go ld was removed from soluti ons co ntaining
AuCl"4 in th e range 2 to 10 mg/L with Azalia
Jiliculoides4 'i . The max imum specific Pb so rpti on by
Rhizapus o/igosporus increased with an increase in
initial lead co ncentrati on up to 200 mg/L beyo nd
whi ch the system attained equilibrium 82 . Simil ar has
been reported fo r sil ver bi oso rpti on by mi cromycete91 .
In co ntrast to th e above reports , S ai and Abraham88
have observed a decrease in percentage adso rpti on of
Cr ions by Rhizapus nigricans with an increase in the
initial metal co ncentrati on from 50 to 400 mg/L.
However, they have reported an increase in the
spec ific biosorpti on with an increase in ex tern al metal
concentrati on.
Biosorbent concentration
With an increase in th e bi osorbent co nce ntrati on
there res ul ts a corres pondin g increase in the total
metal removal acco mpani ed by a decrease in th e
specific uptake. This is du e to th e fac t th at total
adsorption is dependent upon th e number of ava il able
binding sites whereas specifi c uptake is calcul ated as
the amount of metal adso rbed per we ight of the
biosorbent l,92 .
953
Simil ar has been reported fo r so rpti on of Cu, Fe, Ni
and Zn by immobili zed biomass of Phormidium
laminosum72 . A reverse trend was observed in case of
immobili zed li ve cells of Rhizopus oligosporus where
there was an increase in the specifi c bioso rpti on with
increase in bi omass 69 . The authors however observed
a dec rease in the spec ifi c biosorpti on with increase in
biosorbent concentrati on in case of free Rhizopus
oligosporus cells.
A decrease in specific uptake by encapsu lated
bioso rbent with an increase in th e loadin g capac ity of
the bi osobent was reported for A ureobasidium
pullulans 59 . An opti mum biomass conce ntrati on of
5 mg/L res ul ted in 99 .9% removal of go ld by Azolla
f iliculoides45 . Sil ver removal increased with bi omass
co ncentrati on at 1 mg/cm3 to 25 % and at 8 mg/cm 3 to
78 %84 . For all initi al lead concentrati ons studi ed , the
maximum specific lead uptake decreased with
increasing bi osorbent co ncentrati ons2 . They obtai ned
max imum lead uptake capac ity of 126 mg/g with an
initi al lead to bi oso rbent rati o of 750 mg/g. Simil ar
res ults of decrease in specific metal uptake with
increasing biomass co nce ntrati on has also been
reported for Pb, Cd and Zn so rpti on by Citrobacrer
strain MCM S-1 8 138 . Zhou93 observed a decrease in
Zn bi oso rpti on with increase in biosorbent (Rhizopus
arrhizus) part ic le size and its co nce ntrati on. Bai and
Abarh amR8 also reported simil ar trend fo r removal of
Cr ions by Rhizapus nig ricans.
Presence of cations, anions and ligands
Cations
Ca ti ons decrease the bioso rpti on of a metal by
co mpetin g fo r the sa me metal bindjng sites. Thus,
effect of cati ons on biosorpti on can lead to th e co nstru cti on of selecti vity of bi osorpti on seri es based on
biosorpti on of metals from a mi xture solution. For
bi osorpti on there exi sts no co mpetiti on between hard
and soft metals but co mpetiti on among boderlin e metals occur due to simjlariti es in their co-ordin ati on
2
.
Cheml stry .
Metal so rpti on by Cilrobacter strain MCM B-1 8 1
bio mass in presence of equimolar multimetal cations
at pH 4.5 and 6.0 revealed a preferenti al order of
metal sorpti on as Co < Ni < Cd < Cu < Zn < Pb at p H
4. 5 and Co < Ni < Cd < Cu < Zn at pH 6.0 38 . Sar et
al.35 have in ves ti gated the effect of the presence of cocati ons on Ni and Cu sorpti on in Pseudomonas
aeruginosa they have reported th at the presence of Cr
and Zn marginall y dec reased Ni sorpti on whil e ell
954
INDIAN J EXP BIOL. SEPTEMB ER 2003
sorpti o n was o nl y sli g htly inhibited by Zn. Fe, Cu, Co
and Pb caused so me deg ree of inhibiti o n in Ni
sorp ti o n as was observed for Cu so rpti o n in presence
of Fe and Co. Cd was th e most po ten t inhibitor of Ni
sorpti on as was Pb for Cu sorption . Sorption of both
the meta ls was enh anced in presence of Na, K and Ca.
Radi oacti ve e leme nts can be strongly bindin g and
therefo re radi oacti ve and heavy me ta l io ns mutu all y
infl uence eac h other's upta ke . Sar and D 'So uza 3? in
case of Pseudomollas strain observed a sig nifi cant
antagon ism in U sorpti o n to be offered by Th , Fe, Al
and C u whi le meta ls like Cd, Pb and Ag had no effec t.
Soft metal like Au , Ag have a cova le nt bindin g
tendency, th e co mpetition by e lec trostati c bindin g of
other anions could no t be severe for thi s metal. For
competiti on in covale nt bindin g the charge of th e
metal is irre levant. The level of co mpetition depe nds
mainl y o n whether two meta ls use the sa me binding
s ites and how stron g ly they are bo und to any g iven
site. The effect of co mpetin g io ns (Cd and Na) o n
si Iver sorption by Saccharomyces ce revisiae was
84
st udi ed by Singleton and Simmon s . They o bserved
that Cd inhibited Ag+ sorption by 21.2% at 10 mmol/dm'
while Na+ affected biosorpt ion w he n 1000-fold in
24
excess o f Ag in so luti o n. Say el al. observed lowe r
co mpetitiv e bi osorpti o n capac iti es fo r Cd , Pb and Cu
by Phaenerochaele chrysosporium as co mpared to
those unde r non-competiti ve cond iti o ns . The biomass
fo ll owed the a ffinity seri es Cu > Pb > Cd.
Anions alld ligands
A ni o ns of the meta l sa lt balance th e positive c harge
of the meta l io n and occur in a ny meta l bearing
so luti on. Th e most impo rtant effect of anions on
biosorption is thro ugh the formation of comp lexes
with metal ion s in so luti o n. Wh eth er thi s has a
pos iti ve o r negative effect o n the overall metal
sorption depends on the leve l of affinity of the
biomass for th e co mplex in comparison to th e free
94
meta l cation . Greene el al. o bserved the deg ree of
gold so rpti o n to be strong ly depende nt o n th e
compet in g li ga nd s present in th e so luti o n. The anion s
ca n also affec t meta l so rption process by binding to
the ac ti ve site and changing their ch arge.
38
Puranik and Pakn ikar whil e investi gatin g meta l
sorpti o n by Cilrobacler stra in MCM 8-1 8 1 observed
a decrease in the ra nge 0.7-15 % for meta l so rption in
presence of equimolar co ncentrati o n o f borate,
carbonate, chl orid e, s ulfate, nitrate a nd acetate while
in presence of ph osphate and c itrate the uptake of the
meta ls was reduced in the ran ge 24-86 % . Amo ng th e
anions tested for th e ir effect o n U sorption by
Pseudomonas strai n, Sar and D 'So uza 3? observed
18% and 26 % reduction in U uptake by for
lyophili zed and li ve ce ll s, res pect ive ly in presence of
CO/". Zn sorption by Rhizopus arrhizus was reduced
in presence of ligands chiefly du e to formation of
metal co mpl exes of less biosorbabl e nature in the
93
88
seri es EDT A> SO ~- > C1 . . Single ton an d Simmons
studied the effect of li gands (S O ~ - a nd methionin e)
on sil ver sorption by Saccharomyces cerevisiae. They
observed th at methionine inhibited Ag+ sorpti o n by
3
! 3.3% at 100 mmolldm while SO ~ affected biosorption when present in lOO-fold excess of Ag in the
95
so luti o n. T obin et al . have reported the inhibitory
seri es of anions fo r metal sorption by Rhizopus arrhizus
biomass to fo ll ow the order EDTA »SO ;- >cr>
PO ~- >g lutamate>CO i- .
Temperature
Simpl e phy sica l sorption is generally exothermic.
However thi s cannot be extrapo lated to the
phe no menon of bi osorpti o n. The firs t and foremost
reason bei ng, biosorption basicall y inv o lves an
exchange of two metal s . Thu s, the overall reac ti o n
can be e ither e ndo or exoth ermi c. Th e e nergy
liberated due to the binding of o ne ion is compensated
by absorption of the same by the io n re leased.
Second ly, apart from ion-exch ange th e phenomenon
in vo lves compl ex fo rmation as well. As biosorption
occurs in a ve ry narrow temperat ure range (5 ° to
40°C) temperature e ffect s are o nly of secondary
importance.
Go ld sorpti o n by Azolla fiheuloides was found to
be independe nt of te mperature within the range
1O_50°C45 . Suhasini el al 85 observed a decrease in Co
sorption with inc rease in te mperature fro m 190 mg/g
at 30°C to 168 mg/g at 45 °C by th e bi osorbent PFB I .
Sim il ar reports of dec rease in Ag so rpti o n by
Saccha romyces eerevisiae from 185 /-1mo l Ag/g dry
weight at 4°C to 168.4 /-1mol Ag/g dry weight at 55 °C
84
has been reported by S ing leton and Simmons .
38
Puranik and Paknikar have observed no significant
difference in spec ific metal upta ke by Cilrobacler
biomass in the te mpe rature range 4° to 55°C.
Optimum silver so rption by mi cro myce te was
Y1
observed in the te mperature range 30° to 50°C •
Upo n a decrease in te mperature to 6°C o r an increase
to 80°C th e level of sil ver ex tracti o n reduced by
88
10-20%. B a i and Abraham have re ported hi gher Cr
remova l efficiency by Rhizopus nigrieans biomass at
GUPTA & MOHAPATRA : MICROBIAL BIOMASS FOR REMOVAL OF HEAVY METALS
temperature above 30°C; nevertheless there was a
decrease in sorption at higher temperature of 50°C.
They attributed this dec rease to the possible damage
caused to the active binding sites in th e biomass.
Pre-treatments
As biosorption process involves mainly cell surface
sequestrati on, cell wall modifi cation can greatly alter
the binding of metal ion s. A number of methods have
been employed for cell wall modific at ion of microbi al
cells in order to enhance the metal binding capacity of
biomass and to elucidate th e mechanism of
biosorption . These modificati ons can be introduced at
two stages: either durin g growth or to the pre-grown
biomass.
During the growth of microorgani sm the condition
in which a microorganism is grown ca n determine the
biosorption potential since it affects the cell surface
phenotype96 Many reports exist relatin g to the effect
of culture conditions of cells on the bi osorpti ve
. o f' yeast an d f un gl·97 ·'18 . However, not muc h .IS
capacity
known about th e changes in biosorptive capacity of
alga l biomass and cyanobacterium . In Phormidium
laminosum , biosorpti on dec reased initi ally with
nitrogen starvati on and subsequentl y increased until it
reached the va lue of nitrogen suffici ent cellslJ9
In the pre-grown biomass several physical and
chemical treatments have been tried to tailor the metal
of biomass
to
specific
binding
properti es
requirements JOo . The physical treatments include
heatin g/boiling;
freezing/thawing ,
dryin g
and
lyophili zation while th e chemical treatments invol ve
the use of alk ali , acid, organic solvents and salt
solutionsl<l l. Sar el al. 35 have reported enhancement of
both Ni and Cu sorpti on by alkali treatment (NaO H
0. 1 mM) in case of Pseudomonas aerug inosa. Acid
treatment with HCI (0. 1 mM) did not stimulate
sorption whereas autoclaving and oven heatin g
sli ghtly inhibited th e sorpti on. Among organic
chemical s tested tolu ene and ethanol sli ghtl y
enhanced sorption in co ntrast to acetone which
inhibited metal so rption . Meth anol - chloroform
mixture did not alter metal sorpti on whereas lysozy me
pretreated biomass showed reduced sorption of both
th e metal s. Most severe decline in metal sorpti on was
observed following pre- treatment with SDS. Puranik
and Paknikar38 have reported 13-68% decrease in Pb
sorption du e to heat and chemical treatment and
22-78% decrease in Zn sorption after most other pretreatments in case of Cilrobacler strain MCM 8-1 8 l.
In contrast to these, pre- treatme nt of th e biomass with
955
NaOH, Triton X-IOO, Na2C03, (NH 4h S04, ethanol,
methanol and acetone resulted in 37-109% increase in
Cd uptake. Treatment of Saccharomyces ce revisiae
biomass with NaOH or SDS at lOO°C for 30 min
decreased the Ag biosorpti on capacity of th e bi omass
4
to 43 .5% and 70.9% respectivell . However, no
other treatments (HN0 3, (N H4h S04 or EDT A) has
any effect on Ag biosorption. Na2C03 and ethanol
follow ed thi s while H2S04 had an adverse effect on
biosorption . Pre- treatment of Sargassum bi omass with
Ca, Na, Mg and KOH re vea led that KOH washin g
res ulted in stabl e biosorbent with improved affinity
for Zn 102. The stability of th e bioso rbent following
KOH treatment can be attributed to less loss of
organi c carbon du e to the treatment while ac id
treatment caused much damage to the biosorbent and
res ulted in greater loss in total organic carbon.
Streptomyces rilllosus biomass pre-treated with NaO H
(1 mol/dm 3) was able to bind more Zn than the
untreated one lO3 . Baik et al.79 observed that pretreatment of Aspergillus niger, Rhizopus oryzae and
Mu cor rouxii with NaOH (4M) at 121 °C enh anced
drasticall y the sorption of Cu, Cd , Ni and Zn as
104
co mpared with the untreated biomass. Fourest el 01.
have reported cationi c activation of metal sorption
following saturation of Rhizopus arrh izus, Muco r
miehei and Penicilliwn chrysogenum biomasses with
Ca.
Mechanism of biosorption
Sound understandin g of the chemical nature of the
metal ions and the binding sites is imperati ve and
indi spensable to develop an understandin g towards
th e mec hani sm .of biosorption. On th e other hand,
co mpl ex nature of th e biopolymers poses a hindrance
in und ersta ndin g th e mechani sm of the biosorption.
Preliminary studi es carried out on bioso rpti on reveals
it to be a complex interplay of the properties of th e
biomolecules of the cell wall and th e chemical nature
of the metal ion in question . Each of the microbial
group is characterized by a di stinct cell wall structure.
Biosorption by these microbes is attributed mainly to
the li ga nd s present in th e biomolecules of their cell
wall polymers. Hunt l05 ha s elucidated the role of
various biopolymers in metal biosorption.
Binding sites
The cell wall polymers provide a multitude of
chemical groups such as hydroxyl, carbonyl ,
carboxyl, sulfhydryl, thioether, sulfonate, amine,
Imine,
amide,
imidazol e,
phosphonate
and
INDI AN J EXP BI Ol, SEPTEMB ER 2003
95 6
phosphodi esterl 5 . These chemi cal gro ups of the
biopolymers in turn harbor th e binding sites, whi ch
provide th e ligand ato ms to fo rm co mp lexes with
·
105 .106 . T a bl e 3 I'Ists varIOUS
.
meta I b'111 d'll1 g
meta I Ions
groups in bi ological polymers. Eac h of the binding
sites ca n parti cipate in differe nt binding mec hani sms
such as compl exati on and electrostati c attraction of
metal cati ons. Consequently several mechani sms act
in combinati on. In general for metal binding we can
distin gui sh between ion exc hange, so rption of
electri call y neutral materi al (solubl e metal ligand
co mpl exes)
to
specific
binding
sites
and
microprecipitation. These main mec hani sms are based
on sorbate-solve nt interactions, which in turn rely on
so me comb inati on of covalent, elec trostati c, and Van
del' Waal's forces.
Importance of the give n gro up for biosorpti on of
certain metal by certain biomass depends on various
fac tors such as (i) qu antity of sites in the biosorbent
materi al (ii ) chemical state of the site (i.e. its
ava il ab ility) (iii ) access ibility of the site (iv) affinity
between th e site and th e metal (i.e. binding strength ).
For covalent metal binding eve n an already occ upi ed
site is theo reti call y ava ilable. But to what ex tent thi s
site ca n be used by the metal ion for binding depends
on the bi ndin g strength and co nce ntration of th e metal
in co mpari so n to th at already occupying th e site. For
electrostati c binding a site is ava il ab le onl y if it is
ioni zed 15 .
Metal affinity towards biomolecules
The affinity of vari ous metal vari es with respect to
the bimo lec ular ligand. The bond character in
biosorpti on can be explained parti all y by Pearson's
lo7
co ncept of hard and soft metallic ions . Thi s scale is
based on the binding strength of the ions with F and
Tabl e 3 -
r. Those metallic ions (e.g.
Na, Mg, K, Ca etc.) whi ch
form strong binding with F- are referred to as ' hard '
while those forming relatively weaker bonds (e.g. Au,
Ag, Pb, Hg etc.) are referred to as 'soft' ions. A ve ry
and Tobin lo8 have studi ed the applicabi li ty of ' hard
and soft ' principle in predictin g metal so rpti on by
Saccharomyces cerevisiae. There also exists a class of
ions with intermedi ate degree of hardn ess (e.g. Zn,
Cu, CO, Ni, Fe etc.) and are refen'ed to as ' transition'
metals. The hard ions also serve, as essenti al
bi ologica l nutri ents whil e th e soft ions are usuall y
tox ic in nature, The transition metals of intermedi ate
hardn ess are less toxic and are present in certain
biomolec ul es where they ass ist in medi ating spec ifi c
bi ochemi cal reacti ons 109 . Among the ligand atoms '0 '
and 'F' are sonsidered hard , 'S and ' P' are considered
soft while 'N' stays in the intermedi ate category .
The hard ions in biological system fo rm stable
bonds with hydroxy l, phosphonate, phosphate,
carboxy l and carbony l group ; all of which contain '0 '
atoms. The soft ions on the other hand from very
stro ng bonds with sulfhydryl, amine, imidazo le,
amide and imine gro ups i.e. groups ri ch in Sand N
atoms. The hard ions main ly demon strate ioni c
binding whi le th e soft ion di spl ays a coval ent
character l09 ,
Ni eboer and Ri chm'dso n 110 have classified the
metal ions into class A (0 seeking), ' class B (N/S
seekin g) and class C (boderline or intermedi ate
character), based on their binding preferences towards
the 0 , N or S containing ligands of biomolec ul es,
Overall mechanism of biosorption
As mentioned earlier the phenomenon of biosorpti on
may inc lude a combination of several mechanisms
such as electrostatic attraction, complexati on,
Meta l binding groups in bio log ica l poly me rs
C he mi ca l group
Structu ra l form ula
Occ urrence in biomolecu les
Hydrox y l
Carbo ny l (keton e)
Ca rboxy l
Su lfh yd ryl (thiol )
Thi oeth e r
Su lfona te
Am in e
Seco ndary amine
Imin e
A mid e
Imidazole
Phosphonate
Phosphodieste r
-O H
>C=O
-COO H
- SH
>S
Po lysacc harides, uroni c aci d and a min o ac ids
Pe ptides a nd prote in s
Uroni c ac ids, amin o ac ids
Amino ac ids
Amin o ac ids
Sulfated pol ysacc harides
C hi tosa n, am in o acids
C hitin , pe ptidog lyca n, pe ptide bonds
Amino acids
A min o acids
Amino ac ids
Phospholipids
T e icho ic ac id, lipopol ysacc haride
-N H2
>N H
= NH
-CON H 2
-P-2(OH)=O
>POO H
li ga nd atoms
0
0
0
S
S
0
N
N
N
N
N
0
0
GUPTA & MOHAPATRA: MfCROBIAL BIOMASS FOR REMOVAL OF HEAVY METALS
+
ion-exchange, covalent binding, Van der Waal's
d·
.. . III 11 3
.
forces, a d sorption an mlcropreclpltatlOn . .
Complex formations in general involve both
covalent and electrostatic components, whose relative
contribution can be estimated by knowing how
specific the binding is. In case of electrostatic
attraction the binding strength correlates directly with
the charge density (Z2/ rhyd). This implies that the ion s
of the same charge (z) and hydrated radiu s (rhyd)
should be bound with equal strength. A major
deviation from thi s ~inding strength indic ates a
l5
tendency towards covalent bond character . Knowledge
about the ion s re leased provides information about the
I
have reported
bond type. Yang and Volesk/
biosorption of Cd to be accompanied by the release of
hydrogen protons from protonated non-living biomass
of Sargassum jluilans . They observed that the uptake
of Cd and rel ease of protons matched throughout the
84
biosorption process. Singleton and Simmons
observed increased release of H+, Mg2+ and ci+ with
a corresponding increase in Ag biosorption by
Saccharomyces cerevisiae . In general electrostatically
bound ions cannot di spl ace covalently bound ions. As
observed in certain cases proton release occurred only
during heavy metal uptake and not during light meta l
uptake. As protons are bound covalently, these heavy
metals mu st have bound more covalently than th at the
light metal ions.
Similarly inhibition of metal sorption by Na, Ca or
Mg implies greater electrostatic binding of these ions
l1 4
in comparison to other metal ion s . As mentioned
earlier concomitant release of Ca and Mg ions during
Ag sorption by Saccharomyces cerevisiae has been
84
observed . Reports of release of monovalent and
divalent ion s during biosorption has also confirmed
by instrumental analysis82.115 .
Apart from the above mentioned mechanisms there
exists the
phenomenon
of
' adsorption'
and
' microprecipitation ' which describes the accumulation
of electrically neutral metal ions without the release of
any other bound ion27. The phenomenon of adsorption
is driven by the affinity between the sorbate and the
sorbent whil e in case of microprecipitation it is driven
by the limited so lubility of the solute in the solvent. A
less hydrophilic molecule has lower affinity for the
liquid phase and consequently gets adsorbed more
5
easil/ . Microprecipitation of the metal cations and
anions which are often the metabolic products of
certain biomass types form insoluble aggregates (as
salts or complexes) such as sulfides, carbonates, oxides,
oxalates and
phosphonates82. 11 6-11 8. Tran smi ssion
957
electron microscope studies carried out on Rhizopus
oligosporus revealed the deposition of Pb only on the
11 8
82
s urface of cell wa1l . Figueira el al.
have rep0l1ed
2
3
the complexation of Fe + and Fe + with sulfate and
carboxyl groups of Sargassum biomass. Swift and
Forciniti 11 7 observed the deposition of lead as lead
phosphate prec ipitate in the vicinity of the cell wall of
Anabaena cylindrica. The pH of the system also goes a
long
way
in
influencing
the
process
of
.
. . . 58 .119. G reene et aI . PO
- h ave reporte d
mlcropreclpltatlon
interference of HC0 3- in a pH dependent manner
during U sorption to Chlorella vulgaris.
During ion-exchange mechanism of metal sorption
the charge of the ions taken up by the bioso rbe nt
equals the charge of the ions released. This results in
maintaining the neutral charge of the biosorbent. The
acquisition of these charged ions in turn can occur
through a variety of other mechani sms such as
electrostatic attraction or complexation . Regardless of
the mode, the main driving force behind the
mechanism is the attraction of the metal ion for the
biosorbent.
Discerning the groups involved in biosorptioll
One of the major challenges in knowing the
chemical groups involved in biosorption is the
complex nature of the microbial biosorbent material.
Techniques s uch as modification/blocking of
chemical groups have been used for indirectly
deducing the mechanism of bioso rption . Ashkenazy et
121
al.
investigated Pb biosorption of acetone washed
yeast biomass by chemical modifications of the cell
wall components. Propylamine was used for parti al as
well as complete acetylation of hydroxyl and am ino
groups. Also, amino group modifications were carried
out by reacting these groups with tri-nitrobenze ne
sulfonicacid
(TNBS)
and
aminosuccinylation
reaction. The authors observed an increase in
biosorption following modification of amino groups
while acetylation of the hydroxyl group decreased the
biosorptive capacity. This indicated involvement of
negatively charged carboxyl group and amino group
in Pb biosorption. Carboxyl groups were suggested to
be involved in binding Cu and Al in algal species, as
blocking of carboxyl groups by esterification lead to a
decrease in metal binding l22. In Sargassum nalans, the
functional groups, viz. carbonyl (C=O) and amine
(-NH2) were found to provide binding sites for
metals 123.124. Akhtar el al. 125observed 90% decrease in
metal sorption by fungal biomass following chemical
modification of carboxylic acid functional groups.
958
INDIAN J EXP BIOL, SEPTEMBER 2003
The advent of modern instrumental techniques like
electron mi croscopy, energy di spersive X-ray analysis
(EDAX), infrared spectroscopy (IR and FTIR) and
electron spin reso nance spectroscopy (ES R) has
helped in developing a better understanding regarding
th e in vo lvement of the functional grou ps in the bond
fo rmati on process. Table 4 li sts the analys is carri ed
out to reveal th e groups/li ga nds in vo lved In
biosorption. Electron mi croscopi c tec hniques such as
SEM and TEM are helpful in carrying out the studi es
on locali zation of th e metals ca used due to
precipitation , form ati on of intrace llular complexes.
TEM of Rhizopus oligosporus cell s ex posed to Pb
revealed the deposition of Pb only on the surface of
cell wa ll s82 .
X-ray spec troscopic tec hniqu es are based on th e
principle th at a matter can absorb X-rays, giving ri se
to X -ray absorpti on spectra. The fundamental frequencies observed are charac teri sti c of th e functional
groups co ncerned and are absolutely specific. Thi s
gives ri se to the term fingerprint for infrared pattern
obtained. As -the nllInber of fun ctional gro up increase
in more co mpl ex molecu les, the absorpti on bands in
the IR pattern become more difficult to ass ign. However, in such cases group frequencies ari se that help to
simpli fy interpretati on. These groups of certain bands
regu larl y appear near th e sa me wavelength and may
be ass igned to specific molec ul ar groupin gs. Such
group frequencies are ex tremely valuab le in structural
diagnosis . The freq uency associated with a parti cul ar
group vari es sli ghtl y, owing to the influence of th e
molecular environmen t. Such vari ati ons are extremely
useful in structural bioc hemi stry studi es where th ey
he lp to di stin gui sh between bond vibrat ions of different chemi cal groups. For exampl e the C-H bond
variatio n in meth ylene (-CH 2) and meth yl (-CH3)
group, C=O vari ati on in carboxy l (-COO H) and ca rbo nyl (>C=O) group . A decrease in wavelength also
occu rs when double bonds are formed as the stretching frequency increases. FTIR technique has been
widely used for di scerning th e functiona l groups in vo Ive d ·In b'loso rptl.on58 .12) .124. 127 .
The energy di spersive X-ray spectroscopy anal ys is
is based on the princip le th at X-rays can be absorbed
by matter, which gives ri se to X-ray absorption
spectra . These X-ray di spersion spectra may be
detected at various angles that can th en be co-related
with th e complex formed. EDAX anal ys is of
Pseudomonas aerug inosa ex posed to Cu revealed it to
be acc umul ated principall y as CuS, Cu)N, Cu)p and
CuO I26.
The electron spin reso nance spectroscopy (ESR )
employs the mag netic phenomena of the charged
parti cles. Thi s mag neti c phenomen on in molecu les
ari ses due to th e spin of the charged parti cles i.e.
electron s. Energy is requi red to cause thi s resonance
co nditi on, whi ch is abso rbed and recorded as peak in
th e ESR spectrum . Th is indi cates the prese nce of
para magneti c species. The area un de r th e peak is
proportional to the concentration of th e species.
Calibration of the ins trument with known standa rd s
all ows th e co ncentration to be calculated. Philip e l
al. 11 5 usin g thi s techniqu e have reported the '0' group
of ca rboxy l peptidoglycan and ' N' of am inosuga rs or
structural protein s to be assoc iated with Cu binding
on Bacillus polymyxa .
X-ray photoelectro n spectroscopy fo r chem ical
analys is (ESCA) or simply X-ray photoelectro n
spectroscopy (X PS) is a relatively new technique fo r
determ inati on of binding energy of electron s in
ato ms/molecul es, whi ch depends on di stributi on of
vale nce charge and thus gives inforlllation about the
ox idat ion state of th e atom/ion. Pethkar et al. 127 using
thi s tec hniqu e have reported the in volvement of' ,
ato m
in
Ag
sorption
by
Cladosporium
cladosporioides sp.2 at hi gher pH .
Mathematical considerations in biosorption studies
Application of adsorption isotherms in biosorption
studies
The biosorptive meta l uptake of an organism can be
qu antitati vely evaluated fro m ex perimental biosorption
equi librium isotherms similar to those used for the
performance evaluat ion of acti vated carbo ns. The
graphi cal expression is a plot of th e specifi c metal
uptake (q) by the biosorbent against the residua l metal
concentration. The resulting plot is oflen hyperbolic
with the uptake va lue often reac hing a statio nary phase
as it approach's saturati on at hi gh co ncent rati ons of the
adsorbate. The two wide ly accepted and lineari zed
adsorption isotherm models used in the literature were
those proposed by Langmuir l29 and Freund lich 130 and
being named after them.
The general form of the Langmuir model equ ati on is
q =qobC/l + bC
where q
specifi c uptake at residual/equilibrium
co ncentrati on
max imum uptake
qo
residu ai/equi li brium concentrati on
C
constant
rel ated to energy
of
and
b
adsorption
GUPTA & MOHAPATRA : MICROB IAL BIOMASS FOR REMOVAL OF HEAVY METALS
Tabl e 4 -
t
U~e
959
of instrumenta l analysis for determinin g/predi c tin g c he mi ca l gro ups involved
Organism
Analysis
Bonds/Gro ups invo lved
Refere nces
Pseudomonas aerugillosa
(E DAX )
Cu acc umu lated as CuS. Cu ) N, Cu )p and CuO
Ganodenna lucidium
E PR
'0' dominating atom in binding
31
Bacillus polYlllyxa
EPR
'0' of carboxyl group of pe ptidog lyca n and 'N ' of
aminos uga rs o r struc tu ra l prote ins to be associated with Cli
binding
II S
Anabaena cylilldrica
EDAX cou pled with S EM
a nd TEM
Pb de posited as polyphosphate bodies
11 7
Rhizopus o/igosporus
TEM and EDAX
T EM studi es revealed deposition of Pb o nl y o n th e su rface
of ce ll wa ll. EDAX revea led disappearance of peak s
co rrespondin g to Mg2+, S2+, K+ a nd appearance of Pb 2+
along with existing p+
82
Aspergillus niger. Penicillium
chrysogenu11l, Mucor miehei
FT IR
Uranyl binds to amine and amide sites . UO vibration band
appears at a wave number which varies according to pH
and nature of the metal io n spec ies in so luti o n
S8
Chlorella vulgaris
XANES (X- ray Absorption
Near Edge Structure)
Au was bound in + I ox id ati o n sta te i.e. reduction occu rred
from Au 3+ to Au 1+. Under hi gh conce ntrati o n of AlICI ),
red uct io n was pa rti al.
20
EXAFS (Ex te nded X-ray
Absorption Fine Structure)
AuCI ) is bound in co-ordin ati o n with 'N o r '0' ato ms
(EXAFS cannot distinguish be twee n nearest ne ig hbo rs
in a periodic table). In AuCI 2 the principle binding ato m
was'S '
Sargassum IWlans
ESC A o r XPS a nd FT IR
ESCA revea led th e deposition of go ld in e lementa l form
(AuO) whi le FTIR indi ca ted the invo lve me nt o f ca rbo ny l
(>C=O) a nd amino gro ups in the me tal binding.
123
Pseudomonas ael'llginosa
EDAX
Lan tha num uptake by d isp lace me nt of Ca & Mg
36
Rhizopus spp. Based biosorbent EDAX and EPR
EDAX revealed rep lace me nt of Ca ions by Co while EPR
showed th e invo lvement of free orga nic rad ica l ( not
spec ifi ed)
8S
Rhizoplts oligosporus
T EM a nd EDAX
TEM revea led e lec tron dense a rea o n the middl e sec ti o n of
the ce ll wal l following ex posu re to Cd. EDAX s pectrum
co nspicuo us P and Cd peak indi catin g precipitation of
insoluble metal lic Cd as P0 4-3 precipitate
69
Cladosporium cladosporioides
XPS and FTIR
Strain 1: XPS revea led that at low pH 2.0, 'C' and '0'
group not involved in gold binding.
127
126
Binding of Au was accompanied by appearance of 'N- .
Hi g he r pH 7.0 sho wed presence of 'C', '0' , ' N' ~ II of
which participated in Ag bi oso rpt io n.
Strain 2: Gold inte racts with ' 0 ' and ' N' peak while Ag
reac ted with the 'N' g ro up only.
Strain 1: FTIR ana lys is revea led unmaski ng >C=O and COO H gro up which did no t chan ge eve n afte r exposure to
Au , implying binding of Au as AUCI"4 anion at pH 2.0.
In vo lveme nt of CoO a nd C=O in Ag sorpti o n.
Strain 2: CoN a nd CoO were probabl y involved in AU
binding . C-H, N-H_ O-H, CoO a nd NH 3+ a ll were in vo lved
in Ag sorption.
Pseudomonas aerugill osa
IR and EDAX
X-ray diffraction revealed th e prese nce of Cu as CuO as
we ll as CuS while Ni was present as P, Nand C.
3S
FTIR revea led the precipitation of carboxy l, carbo ny l a nd
phophoryl groups along with H-bo nding in metal so rpti on.
Chi orella fus ca
X-ray spec troscopy
Cu sequestered as po lyphos ph ate bodies
128
INDIAN J EXP BIOL, SEPTEMBER 2003
960
Taking the inverse of both sides, the equation can
be linearized as follow s:
IIq = I/Q l11ox .b.C + IIQl11ax
The Freundlich adsorption model has the general form
l /n
q=k C
Thi s can be lineari zed by taking the natural logarithm
of both sides of th e equ ation to give,
In q=ln k+ lin In C
The intercept In k gives the meas ure of adsorbent
capacity while the slope lin gives the intensity of
adsorption.
The Freundlich isoth erm is more preferred for
aqueo us solutions whi le · the Langmuir isotherm is
preferred more for gaseous solutions. Though both th e
models desc ribe biosorpti on data well, but they are
not models whose terms and parameters would have a
co nveni ent and appropriate physical interpretation
attac hed to them I. An isotherm th at is steep at the
origin at low residual co ncentrations of the sorbate is
highl y desirable because it indicates a hi gh affi nity of
the sorbent for the given sorbed species. Such a
biosorbent would be performin g well at very low
co ncentrations of the sorbed species in the solution .
Blanco el al. 72 have reported hi gh Langmuir
constant (Ql11ax) values of 19.53, 17 .S3, 16. 10, IS.05
mglg for Cu, Fe, Ni and Zn res pectively for
polysulfone immobili zed Phorl1lidiul1L laminosum
while that of th e epoxy immobili zed beads were
2 1.2S, 16. 7, 13. 13 and IS.60 for the respecti ve metals.
The binding energy values ran ged between 0.06 for
Ni to 0.36 for Cu in case of polysulfone immobili zed
beads and between 0.10-0.26 for epoxy immobili zed
beads. Sar el al. 35 have evaluated the Ni and Cu
so rpti on capacities of lyophili zed Pseudomonas
aem ginosa cell s using Freundlich isotherm. They
observed th at the sorpti on intensity In k for Ni (0.69)
was hi gher th an th at of Cu (0.52) while the sorpti on
in tensity ( lin ) of Ni (3. 14) was lower than Cu (4.2),
impl ying that in Ni biosorption equilibrium metal
co ncentrati on pl ays an important role as compared to
that in Cu. They also observed that at lower
equilibrium concentration (Ce 20 mg/L ), Ni and Cu
removal capacity was almost the sa me whi le at higher
co ncentration (Ce 200 mg/L) , N i (121.5 mg/g ) was
preferred over Cu (66 mg/g ).
Rai el al. 13 1 ha ve compared the biotechnological
potenti al of laboratory grown and naturally occurring
Mic rocyslis for Ni and Cd biosorption using
Freundlich, Langmuir and BET isotherms. Freundli ch
and Langmuir constants revealed hi gher affinity for
Cd than for Ni. High R2 values of BET isotherms
suggested a mul tiplayer binding of metals. Puranik
and Paknikar38 have obtained hi gh co-relation coefficients for Pb, Cd and Zn biosorpti on (0.99, 0.99
and 0.95, respectively) by Citrobacter strain MCM BlSI using Langmuir isotherm . Highest Qmox va lue of
5S .78 mg/g biomass with b of 0.11 was obtained in
case of Pb while lowest QIll"X= 23.62 mg/g and b of
0.16 was obtained in case of Zn . Sar and D ' Souza 37
obtained a good fit of lineari zed Langmuir and
Freundlich adsorpti on isotherm s for live as well as
lyophilized biomass of Pseudomollas . Philip el
cd. J6 obse rved that U so rpti on by Pseudomonas
aerug inosa fo llowed Langmuir and Freundlich
isoth erm s but not BET isotherms. Zhou 93 compared
the Zn adsorptive capacity of six di fferent fungi
(Rhizopus arrhizus, Mu cor racemosus. Mycotypha
africana, Aspergillus nidulans. Asperg illus niger and
Schizosaccharomyces pombe) by Freund lich and
Langmu ir isotherm mode ls. Amongst the fungi tested
R. a rrhizus had highest K and Qlllax va lue of
11.055)J.mol l . n In/g and 213J.lmo]Jg, res pec ti vely.
Korenevskii et al. 9l evaluated th e bi osorpti on
efficienci es of mi cro my cete cultures u ing Freundlich
and Langmuir constants. They obtained low K, va lue,
which were indi cative of hi gh affinity of th e fung al
biomasses for Ag cation s. Two to three fo ld variations
in Ag sorption capacities was observed among all the
mi cro mycete species. Further vari ati ons were not onl y
subj ec tive to species but also to strains of th e same
spec ies 91 . Bai and Abraham 88 observed th at
eq uilibrium data of Cr sorpti on by Rhizopus nigricans
fitted well into linea ri zed Freundlich isotherm mode ls.
Say et al.24 have reported good fit of Langmuir model
to Cd, Pb and Cu so rpti on by filamentous fun gus
Phaenerochaete chrysosporium . Wong et al.132 ha ve
co mpared the Ni sorption ca pac ities of two Chlorella
spec ies - C vulgaris and C miniata. They have
repo rted max imum Ni uptake to be 641.76 and
1367.62)J.g/g biomass for C vulgaris and C minia/a,
respectively . The experimental values followed th e
sa me trend as predicted by the Langmuir adsorption
isotherm model (29S5.07 and 12S2.05 )J.g/g bi omass
for WWl and C vulgaris res pectivel y) . Further hi gh
correlation co-efficient for the regress ion lines
indicated good fit of the model to both the systems.
Lower n value of C miniata ( 1.33) compared with C
vulga ris (1.5 I) also indi ca ted that th e adsorption by
the former was more effec ti ve at low metal ion
co ncentration 132 . Cd biosorpti on by Ca-alginate
immobilized Trametes versicolor fitted both into the
GUPTA & MOHAPATRA: MICROBIAL BIOMASS FOR REMOVAL OF HEAVY METALS
Langmuir and Freundlich adsorpti o n isotherm mode ls
with very hi gh R2 val ues 23 .
Modeling biosorption process with special emphasis
to multimetat situations
The applicatio n of mathematical mode ls to
bi osorption processes apart from giv ing a qu antitati ve
description of the process also aid in optimi zin g the
Secondly ,
mathe mati cal
operating
conditions.
modeling at bench scale leve ls he lp in minimi zin g the
number of ex perimental run s and he lp to pin-po int the
results of spot check ex perime nts IS. Thirdly, modeling
at indu striai sca le becomes indi spe nsab le as it goes a
lo ng way in reacto r des igning and he lp discover
bottlenecks th ereby optimi z in g the operational
condition s and red uc in g the costs in vo lved in hit and
trial run s.
Though both the Freundlich and Lang muir isotherm models predict th e metal uptake as a fun ct io n of
the co ncentratio n of one metal i.e. in the ir basic fo rm
they are suited fo r mo no meta l systems witho ut pH
effects. Thus, they are no t appropri ate for io nexchange systems , w hi ch usua ll y in vo lve mo re th an
o ne meta l spec ies. C ho ng and Volesky l33 have described a mUlti-component Langmuir isoth e rm mode l,
whi ch ass umes a 1: I stoc hi o metry between metal io ns
and binding sites whereby all metals make use of th e
sa me sites and co mpete for them. The auth o rs have
used three dimensional sorpti o n isotherm s urfaces
usi ng the software MATLAB 4.0 to eva luate the twometal sorpti on performance of the bi osorbe nt. In the ir
studi es o n mode lin g of proto n-meta l ion exchange in
34
biosorption , Schiewer and Volesk/ have extended
the Langmuir mode l for predictions of co mpetition
between divalent heavy metal io ns ( 1:2 stoc hio metry ).
They have rendered direct calc ulati o n of meta l uptake
without the in vo lveme nt of any iterati ve calcul ati o ns.
Schiewer and Voleskyl s have discussed mode ls invo lvin g competition amo ng metal cations, pH effects,
and effects of io ni c streng th and e lec trostati c att racti o ns. Further, they have also di scussed in detail qu antitative mode lin g of batch kinetics. Chong and Volesky 135 have eva luated th e extended mUlti-co mpo ne nt
Langmuir model for ternary metal so luti o ns of Cu, Cd
and Zn. They reached the co nc lu sio n that fittin g of the
multi-compo nent Langmuir data to the tern ary data
was sem i-e mpiric al and so me predictions of the behav ior of ternary meta l syste ms were in a reasonably
good agreement w ith the experime ntal res ults and
those derived from binary systems. The use of tri ang ul ar diagram technique was successfull y · impl e-
96 1
men ted for the graphi c representation o f ternary bio. d ata 13S .
sorption
62
Sanchez el al.
investi gated three models to
pro pose the most suitab le equ ati o n to represent th e
sorption data of Cu-Zn system in 3 D space. The first
model produced an eq uatio n with three parameters,
the seco nd and third had fo ur and five parameters,
res pec ti vely. These para meters were eva lu ated using
MATLAB 4.0 progra m . The in vesti gati o ns revea led
th at a ll the three mode ls studi ed co uld make a good
prediction of the metal uptake for the syste m studi ed
with minimum variance. Thus, the c ho ice of the best
model was restricted by look in g for o ne with th e
lowest number of parameters i.e. mode l 1 w hi ch they
used for the constructi o n of 3D biosorption isotherms.
This model was a binary Langm uir type eq uatio n.
Sag el al. 136 had a lso e mpl oyed the com pet iti ve
Freundlich and Langmuir adsorpt io n mode ls to study
the si multaneous bi osorpti o n o f Cr and Fe on C.
vulgaris a nd R. a rrhizus. They reported the
Freundli ch mode l for binary meta l mi xtures to be
sat isfacto ry for mos t adsorptio n eq uilibrium data of
Cr an d Fe io ns o n C. vulgaris, w hil e the co mpetitive
a nd mod ifi ed Lang muir mode ls were mo re suited to
characteri ze competiti ve adsorpti o n of C r and Fe from
binary systems by R. a rrhizus. Sag el 0 1. 137 have
ana lyzed the eq uilibrium data of Pb, C u and Z n
sorpti o n from binary a nd ternary meta l so luti ons using
e mpiri cal competiti ve Freundlich isotherm model.
Thi s isothe rm mode l is related to the indi vid ua l
isotherm para meters and takes into account th e
correction factors.
l 38
M ehta and Gaur
have reported th e use of twodimensiona l conto ur plots using the g raphi ca l
software Sigma Pl o t 2.0 to de pict th e conC UITent
sorption of Ni and C u by ChIarella vulgaris. Thi s
represented a re latively simpl er approach where the
ex te rnal concentration of meta ls were plotted on the X
a nd Y ax is w hile th e co nto ur lin es co rres po ndin g to
Z-axis represented the corresponding metal sorbed.
Swift and Forciniti 11 7 have deve loped a mass-transfer
kineti c model, w hi ch qu antitati ve ly pred icted the
concentratio n of Pb in ce ll s of Anabaena cylindrica as
a function of spati a l dimensions and time. This model
deals with on ly a mo no meta l situ ation .
Recent advance me nt in software technologies has
also co me a lo ng way in developing software that is
spec ific for predicting biosorption pe rformance in
fixed bed reactors. Figueira el al. 102 have tested one
s uch software package - ' IMPACT' , for eva lu atin g
th e bi osorpti o n performance of Sa rgassum biosorbent
962
INDIAN J EXP BIOL, SEPTEMBER 2003
with a metal mixture (Cu, Cd and Zn) in a column.
They have reported that the application of
experimental IMPACT computer software was only
partially successful in exactly simulating the
biosorption column performance.
Elution and recovery of metal
There are generally two fates of the metal laden
biomass; either it is 'ashed' off' (by incineration) or
regenerated (by elution). The first alternative is
preferred where the biosorbent material is supplied as
a waste biomass - cheap and abundant. This reduces
the volume of waste generated. The more desirable
and economic option of regeneration is based on the
selective stripping 'off' of the metal laden on the
biomass by the use of desorbing agents. The
desorbing agents or eluting solutions function by
uncoupling the bonds formed between the metal and
the biosorbent. Small quantity of the eluant results in
hi gh metal concentration in the resulting solution ,
thus making it amenable to easy and economical
extraction procedures. Thus the solid/liquid ratio
(S/L ) is often used to express the efficiency of the
eluant. The so lid represents the amount of the
biosorbent and the liquid represents the volume of the
eluant applied. Hi gh SIL values are desirable for
complete elution 1.22.
Chelating agents, sa lts and alkali solutions proved
to be the best eluants 22 .38.4o.61 .62.7o.91 while mineral
ac ids thou gh elute considerable amount of
metal22.35.39.85. 139, ususall y cause damage to th e
biosorbent. This in turn affects the 'second resorption '
61
36
and subsequ ent resorption cycle . Philip et al. have
reported 95 % desorption of the loaded U from
Pseudomonas aeruginosa using 0.2 M HC!. They
however observed an ad verse effect of the acid on the
viab ility of the cells. This drawback resulted in opting
for citrate buffer (0.2 M, pH 4 .0) as the eluant of
choice with 80% desorption. Similar decrease in
biosorption capac ity of beads has been reported for
eluti on of metal laden beads of Citrobacter biomass
103
have reported that
by 0.1 M HC1 38 . Addour et al.
Streptomyces rimosus biomass regeneration with
0.1 mol/dm 3 HCI resulted in 90% Zn recovery with
20% weight loss.
Howeve r, reports do exists where the use of acid
elu ants did not affect the biosorptive potential of the
biosorbent in subseq uent cyc]es35.60.64.86.87 . Kam a et
al. 60 have reported 0.1 M HCI to be effecti ve in
eluting Cd and Co. They observed 80% Cd binding
ability to be retained up to three cycles while in case
of Co the loss of the binding ability was rapid with
more than 70% of the initial binding capacity being
lost. Suhasini et al.85 have reported relatively high
readsorption efficiencies (>70%) for PFB 1 following
23
desorption with HC] or H 2S04 (0.1 N). Arica et al.
observed no noticeable change in the adsorption
capacities for Cd by Trametes versicolor following
desorption of the metal laden biomass with HCI
73
(10 mM). Yan and Viraraghavan
have reported
comparable readsorption capacities of polysulfone
immobilized Mucor rouxii biomass followin g
78
desorption with 0 .05 N HNO J . Huang and Huang
have enhanced Cu sorption by ac id washed
Aspergillus oryzae while acid washing did not
adversely affect the metal adsorption capacity of
Rhizopus oryzae myceli a. They have thus
recommended acid wash for the dual purpose of
biomass pre-treatment as well as regeneration .
An important aspect of the metal elution is the
selective and consecutive removal of metals from the
biosorbent. Ahuja et al.53 -55 observed max imum
elution of Zn , Cu and Co by EDT A (10 mM), citrate
buffer (0.2 M, pH 3.0) and Na2C03 (1 mM),
140
respectively . Darnall et al.
have reported a scheme
for selective recovery of Cu 2+, Zn 2+, Au 3+ and Hg2+
bound to polyacrylamide immobi lized Chlorella
vulgaris by lowering the pH from 6.0 to 2.0 and
subsequently treating the column mercaptoethanol to
elute Au 3+ and Hg3+.
Ideally speaking several criteria have been laid
down for the choice of desorbing agents. These
include high SIL rati o, high metal concentration factor
(after desorption and before adsorption), high
efficiency, fast kinetics, selectivity, preserv ation of
structural integrity of the biosorbent , economical and
environment friend ly. However, due to selfcontradicting requirements it often becomes difficult
to look for an eluant possess ing all these major
criteria.
Commercial biosorbents
Various types of microbial biomasses have formed
the basis of formulation of new and potent metal
sequestering biosorbents. This is important in the
present day scenario, as there is an increas ing need for
an effective and economical process to remove metal
ions from industrial wastewater and drinking water.
A potent algal biosorbent AlgaSORB ™ has been
deve loped using a fresh water alga Chlorella vulgaris
l40
to treat wastewater . Thi s can efficientl y remove
metallic ions from dilute soluti ons i.e. 1-100 mg/L
GUPTA & MOHAPATRA : MICROBIAL BIOMASS FOR REMOVAL OF HEAV Y METALS
and reduces the concentration of metal s down to 1
mg/L or even below. Calcium and magnesium also
does not affect the so rption of heavy meta ls by
AlgaSORBTM .
Another metal sorption agent AMT-BIOCLAIMTM
(MRA) has employed
Bacillus biomass to
manufacture granulated materi al for waste water
treatment and meta l recovery . This can accumulate
metal cation s with efficient remov al (>99%) fro m
dilute so lution s. It is non-selective and me tal s can be
stripped from it after loading by H 2S0 4 , NaOH or
complexing agents and the g ranules can be
c
?6
regenerated lor repeated use- .
Bio-Fi x biosorbe nt uses biomass from a vari ety of
sources including cyanobacterium (Spiruiina), yeast ,
algae, plants (Lemna sp.) and g uar gums to g ive a
consistent product and immobili zed as beads using
polysulfone. Zinc bindin g to thi s bioso rbe nt is
approximate ly 4-fold higher th a n the ion-exchange
res in s. There is vari abl e affinity for differe nt meta ls
Al > Cd > Zn > Mn and a much lower affinity for M g
and C a. Metals can be e luted usin g HCI or HN0 3 and
biosorbent can be reused for more th an 120
ex traction-e lution cycles.
T wo marine a lgae name ly, Sargassum natans and
Ascophyllum nodosum have been found to have
excellent biosorption capacities for gold and cobalt
respectively , among the photoautotroph s 11.141 .
Conclusions
Biosorpti on
IS
an
economically
feasible;
efficient
technology
for
metal
technically
removal/recovery and can comfortably fit into the
metal treatment processes a nd is eco-friendly in
nature. In spite, of these advantages why has the
biosorption/wastewater treatme nt remained as an
e mbryo nic industry ? This is because not all the
companies,
which
generate
metal
polluted
wastewater, will have the capability or the interest to
do anything other tha n the basic treatment to comply
with the legislati ve. Hence to overco me thi s what is
needed is a series of spec ialists, centralized facilities
which would be capab le of re mov ing meta l from
waste water and regene rating or process ing the metal
loaded so rbe nt and then converting th e recovered
metal into reusable form. Alternatively, if the
bi osorbent used is a waste product, its inc ine ratio n
could be used to produce meta l rich slag.
Lookin g into the eco nomj cs, feasibility in te rms of
scale up and working efficiency as a technology the
microbi al bi osorbent prov ide encouragin g res ults to
963
be utilized in wastewater treatment. What is needed is
an extra moral input from the indu stries generating
metal polluted wastewater to in vest into such clean up
technologies before di scharging their liquid effluents
into the water bodies.
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