Characterization of Acetone-Washed
Yeast Biomass Functional Groups
Involved in Lead Biosorption
R. Ashkenazy,1 L. Gottlieb,2 S. Yannai1
1
Department of Food Engineering and Biotechnology, TechnionIsrael
Institute of Technology, Haifa 32000, Israel
2
Refael: Armament Developing Authority, Department M1,
Haifa 31021, Israel
Received 13 February 1996; accepted 26 July 1996
Abstract: The mechanism of lead cation biosorption by
acetone-washed biomass of Saccharomyces uvarum
was investigated by chemical modifications and spectro-
scopic monitoring of the cell components. Reacting the
carboxyl groups with propylamine, which neutralizes
these anions, considerably decreased the metallic ion
uptake, indicating that negatively charged carboxyl
groups play an important role in lead biosorption due to
electrostatic attraction. After lead biosorption the photo-
acoustic Fourier transform infrared spectroscopy
showed a change in the symmetrical stretch of the car-
boxylate groups of the acetone-washed yeast biomass,
and the X-ray photoelectron spectroscopy oxygen peak
was also found to be shifted. These findings support the
hypothesis that lead uptake occurs mainly through bind-
ing to the carboxyl group. In X-ray photoelectron spec-
troscopy the nitrogen peak decreased after the biosorp-
tion of lead, suggesting that nitrogen-containing groups
are also involved in the biosorption process. Acylation of
amino groups was shown to increase the lead biosorp-
tion capacity. The acylation reaction converts the posi-
tively charged amino group to an amide capable of co-
ordination to lead cations. Deproteination by boiling the
biosorbent with NaOH increased the lead uptake. The
acetone-washed biomass uptake of lead from an aque-
ous solution at pH 5.5 was 48.9 mg/g dry weight. Pure
chitin adsorbed 48.8 mg lead/g dry weight. Mannan iso-
lated from S. uvarum did not adsorb lead at all. Electro-
static attraction of the carboxyl groups and other anions
present in the acetone-washed biomass, and complex-
ation with nitrogen atoms, especially in chitin, appear to
be the main mechanisms involved in lead cation biosorp-
tion. 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:
110, 1997.
Keywords: metal biosorption; acetone-washed yeast bio-
mass; Saccharomyces uvarum; lead binding mecha-
nisms; X-ray photoelectron spectroscopy; Fourier trans-
form infrared photoacoustic spectroscopy
INTRODUCTION
Earlier experiments in our laboratory proved the ability of
beer yeast (Saccharomyces uvarum) acetone-washed bio-
mass to remove metallic ions from aqueous solutions (Yan-
nai and MeshulamSimon, 1992).
Correspondence to: S. Yannai. Telephone: 972-4-8293350; fax: 972-4-
8320742; e-mail: SYANNAI@TX.TECHNION.AC.IL
1997 John Wiley & Sons, Inc.
The aim of the present study was to characterize the
nature and binding mechanism of chemical groups in the
acetone-washed biomass that were responsible for lead cat-
ion biosorption.
Most of the published reports on metallic ions biosorp-
tion were aimed at assessing the metal-binding capacity, and
only a few attempted to elucidate the mechanism of binding.
Tsezos and Volesky (1982a) reported a three-process
mechanism for uranium biosorption by Rhizopus arrhizus,
based on uranium coordination and biosorption by the cell
wall chitin structure and precipitation of uranylhydroxide
within the chitin microcrystalline cell wall structure. Mura-
leedharan and Venkobachar (1990) showed that cupric ions
chemically coordinate to Ganoderma lucidum. They did not
identify the specific groups responsible for biosorption but
concluded that these groups were neither protein nor chitin.
Kuyucak and Volesky (1989) reported that cobalt biosorp-
tion by a nonliving biomass of Ascophyllum nodosum is
predominantly an ion exchange process. They suggested
that the carboxyl groups of the cell wall alginates play an
important role in cobalt binding. An ion exchange adsorp-
tion mechanism is also suggested by Fourest et al. (1994)
who used Ca2+ saturation or pH controlling during the ad-
sorption process to increase the metal uptake capacity.
Doyle et al. (1980), who selectively modified the free
carboxyl and amino groups of Bacillus subtilis, showed that
when amino groups were replaced by neutral, bulky, or
negatively charged groups, the number of sites available for
cation complexing generally increased and introduction of
positive charges onto the cell wall decreased the numbers of
metal binding sites. They revealed that both teichoic acid
and peptidoglycan moieties contributed to the sites available
for interaction with metallic ions.
Crist and colleagues (1988) suggested that the biosorp-
tion of heavy metals consists of two phases: a fast phase
(less than 4 s) is attributed to surface adsorption, mainly
based on anion exchange with the participation of the car-
boxyl groups of uronic acids; and a much slower metal
uptake (2 h) phase represents the diffusion of ions into the
cell structures.
As can be seen from the above studies, the biosorption
CCC 0006-3592/97/010001-10
mechanism strongly depends on the nature of the biosor-
bent. Algae, fungi, and bacteria differ from each other in
their constitution, giving rise to different mechanisms of
metal biosorption.
Strandberg et al. (1981) suggested two different biosorp-
tion mechanisms: one for bacteria (Pseudomonas aerugi-
nosa), which is intracellular biosorption by living cells, and
another for yeasts (S. cerevisiae), which is an extracellular
biosorption. The bacterial biosorption mechanism is based
on metal binding of metallothionein, whereas the yeast bio-
sorption mechanism is based on electrostatic attraction be-
tween the metallic cations and the negatively charged phos-
phate groups on the surface of the cell wall.
The biosorption mechanism depends also on the nature of
the metal in question. Tsezos and Volesky (1982a,b)
showed that there are different biosorption mechanisms for
uranium and thorium. Holan and Volesky (1994) showed a
conspicuous difference between the biosorbent uptake of
lead and nickel.
One example of the different sorption mechanisms is that
the cis position of sulfate esters at C3 and C4 of the branched
fucose residue may lead to the covalent binding of lead only
to one residue, and the same site could sequester nickel by
an ion exchange mechanism.
The above-mentioned earlier findings showed that S.
uvarum, produced in large amounts by the beer industry and
which has a fairly low economic value, exhibits an appre-
ciable metal uptake capacity for lead and mercury. There-
fore, it was considered worthwhile to study the specific
binding mechanism for this particular species for lead,
which is a major water pollutant in many countries. Eluci-
dation of this mechanism may help in upgrading this bio-
mass to make it a better metallic ion adsorbent. It is quite
likely that more than one binding mechanism exists, be-
cause several acetone-washed biomass components may be
responsible for both complexation and ion-exchange inter-
actions. To explore the biosorption mechanisms, it is nec-
essary to identify the functional groups involved in the bio-
sorption process. For this purpose, spectroscopic methods
such as X-ray photoelectron spectroscopy (XPS) and Fou-
rier transform infrared photoacoustic spectroscopy (FTIR-
PAS) coupled with chemical modifications were used in this
study.
In this article the characterization of changes in some
moieties of the acetone-washed biomass surface before and
after biosorption of lead, are reported. In addition, the metal
binding capacity of different fractions of the biosorbent and
the effect of chemical modification of some functional
groups on their lead binding capacity were also investigated.
MATERIALS AND METHODS
Biosorbent
Spent S. uvarum yeasts (Tempo Beer Brewery, Israel) were
used. Preparation of the biosorbent was as described else-
2 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 55, NO. 1, JULY 5, 1997
where (Yannai and MeshulamSimon, 1992). The main
steps were as follows: the yeast cells were first washed with
distilled water, then with acetone, and finally once more
with water. After each wash the biomass was centrifuged,
and the final product was a biomass consisting mostly of
yeast cell walls.
Materials
Chitin (poly-N-acetylglucosamine), a purified powder from
crab shells, was purchased from Sigma Chemicals. Mannan
was extracted from the S. uvarum acetone-washed prepara-
tion as described by Haworth et al. (1937). Protein-free
acetone-washed biomass components were prepared as fol-
lows: yeast cells were boiled for 8 h with a 6% NaOH
solution. The solution was centrifuged; the nitrogen content
of the remaining solids was determined by Kjeldahls
method; and the protein content was assayed by the protein
assay using a Bio-Rad (Richmond, CA) kit (No. I). The lead
uptake capacity of the treated yeast residue was examined in
batch sorption equilibrium experiments (Volesky, 1990).
Lead Uptake
A lead solution containing 100 mg/L was prepared by dis-
solving lead nitrate (Pb(NO3)2) in deionized water. The
batch sorption experiments were performed in centrifuge
bottles, each containing 1.5 g of solids and 200 mL of lead
solution at 25C, and the pH was adjusted to 5.5 using HCl
or NaOH. The final equilibrium pH was about 5.6.
The bottles were shaken for 20 min by a horizontal me-
chanical shaker at a rate of 130 times per minute. Separation
of the solution from the biomass was achieved by centrifu-
gation. Lead in the solution was determined by atomic ab-
sorption spectrophotometery using a Thermo-Jarrel Ash IL
157 instrument. Each experiment was repeated two or more
times.
Cell Surface Analysis
XPS
This technique was used to evaluate the surface character-
istics using an ESCA-Auger system (PerkinElmer Physical
Electronics Division, model 555). XPS spectra before and
after lead biosorption were taken to identify groups partici-
pating in the biosorption. The acetone-washed biomass was
prepared for the XPS analysis by dehydration, first by over-
night lyophilization and then with a mercury vapor pump
for 8 h, at 106 bar. The acetone-washed yeast biomass layer
analyzed was 50100 thick. Description of the XPS tech-
nique and its interpretation is described by Briggs (1990).
FTIR-PAS
This technique was used to elucidate the chemical charac-
teristics relevant to metallic ion sorption by the acetone-
washed yeast biomass. FTIR photoacoustic spectra were
measured with an MTEC PAC 300 photoacoustic detector
mounted in the sample compartment of a Bio-Rad FTS60A
FTIR spectrometer.
The sample cup was purged with helium and measure-
ments were done at an interferometer mirror velocity cor-
responding to a helium-neon laser fringe modulation fre-
quency of 2.5 kHz at 8 cm1 resolution. Each sample spec-
trum was compared to a carbon black spectrum to remove
spectral variations due to the IR source and spectrometer
optics. Dried acetone-washed biomass samples for PAS
were prepared by suspending the biomass in acetone and
decanting the solvent.
This was repeated 4 times and finally the samples were
vacuum dried at room temperature to a constant weight.
PAS is a thermal detection method in which the absorbing
sample is the surface and the IR spectra of the surface is
effectively recorded (Virdrine, 1981).
Chemical Modification of
Acetone-Washed Biomass
The carbodiimide-nucleophile reaction with carboxyl
groups was used to neutralize the negative charges on the
acetone-washed biomass. This was done according to the
procedure reported by Doyle et al. (1980) except that pro-
pylamine was used instead of ethanolamine.
Aminoacetylation was performed in two ways: one as
described by Doyle et al. (1980) and the other was a total
acetylation of hydroxyl and amino groups in acetic anhy-
dride/pyridine (Fieser and Fieser, 1967). Two more modi-
fications of the amino groups were aminosuccinylation and
the reaction of amino groups with trinitrobenzenesulfonic
acid (TNBS) as described by Doyle et al. (1980). After the
chemical modifications, the lead uptake capacity of the
modified acetone-washed biomass was evaluated by batch
equilibrium sorption experiments.
RESULTS
XPS
Figures 1a and b show the X-ray photoelectron spectra of
the acetone-washed S. uvarum biomass before and after lead
uptake. The atomic concentration of nitrogen before lead
uptake was 8.4% of the total atomic concentration (Fig. 1a),
while after biosorption it dropped to 0.7% (Fig. 1b). The
nitrogen peak was shifted from 400.1 eV before lead uptake
to 401.8 eV after biosorption (Fig. 2a,b, respectively).
The oxygen peak was shifted from 533.1 eV before lead
uptake to 535.2 eV after uptake of lead (Fig. 3a,b, respec-
tively).
FTIR-PAS
The photoacoustic IR spectra of the original acetone-
washed biomass preparation before lead uptake and the ac-
ASHKENAZY, GOTTLIEB, AND YANNAI: LEAD-BINDING GROUPS IN YEAST BIOMASS
etone-washed biomass after lead uptake are shown in Figure
4. Examination of the acetone-washed biomass spectrum
after biosorption reveals a change compared to the original
preparation. The absorption band at 1405 cm1 was shifted
to 1375 cm1.
Metal Uptake Capacity of Different Components
of Acetone-Washed Biomass
Deproteination accomplished by a 1-h boiling of the bio-
sorbent with 3, 6, and 40% NaOH brought an increase in
lead uptake, compared to the biosorption of whole acetone-
washed biomass preparation (Table I). Mannan extracted
from the original spent yeasts according to the procedure of
Haworth et al. (1937) showed no lead sequestration at all.
Using pure chitin gave a metal uptake capacity of 48.8
mg/g dry weight (dry wt) at a 5.25 ppm final lead concen-
tration (Table I).
Lead Binding Capacity of Acetone-Washed
Biomass after Modification of Carboxyl or
Amino Groups
The lead ion sequestration efficiency after reaction of ac-
etone-washed yeast biomass carboxyl groups with propyl-
amine is shown in Table II. As can be seen, the metallic ion
uptake capacity of this derivatized acetone-washed biomass
decreased considerably. On the other hand, acetylation and
succinylation of the amino groups improved the lead uptake
capacity of the acetone-washed biomass. However, TNBS
has no effect on the lead uptake by the yeast acetone-
washed biomass (Table II).
DISCUSSION
XPS
The mechanism of lead biosorption by the acetone-washed
yeast biomass may involve one or both of the following:
coordination complexing with the unpaired electrons of the
nitrogen atom of the amino or amido groups (Tsezos and
Volesky, 1982a,b) or ionic interactions with anionic groups
such as carboxyl (Kuyucak and Volesky, 1989). XPS was
used for the characterization of functional groups in the
yeast acetone-washed biomass before and after lead bio-
sorption. The results follow.
Nitrogen
From the XPS spectrum it can be seen that there is a sig-
nificant difference between the nitrogen peak before and
after lead biosorption (Fig. 1a,b). The atomic concentration
of nitrogen before lead biosorption was 8.4% of the total
atomic concentration, while after biosorption it decreases to
0.7%. Hence, lead biosorption is accompanied by a change
in nitrogen binding, providing evidence that nitrogen-
containing groups take part in lead biosorption. These can
3
Figure 1. XPS analysis spectra of acetone-washed Saccharomyces urarum biomass (a) before and (b) after lead uptake (At. Con., atomic concentration).
be amido groups in chitin, or other nitrogen-containing
groups in the peptidomannan, peptidoglucan, or proteins
that comprise the yeast acetone-washed biomass. After lead
biosorption the nitrogen peak exhibited a shift of 1.7 eV
(Fig. 2a,b). This observation is additional proof that nitro-
gen-containing groups in the yeast acetone-washed biomass
are involved in lead biosorption. Another possibility is that
this shift is a result of a change in the acetone-washed yeast
biomass charges after lead biosorption.
4 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 55, NO. 1, JULY 5, 1997
Oxygen
The oxygen peak was shifted by 2 eV after lead biosorption
(Fig. 3a,b). This shift indicates lead interaction with oxygen
pointing at the carboxyl group involvement in lead uptake.
Phosphate
It was found that uranium biosorption by S. cerevisiae is
through electrostatic interaction with phosphate (Strandberg
 Figure 2. High resolution N 1s XPS spectra of acetone-washed Saccharomyces urarum biomass (a) before and (b) after lead uptake.

et al., 1981). Phosphate peaks were not found in the ac-
etone-washed biomass outer layer of our preparation, as
judged by the XPS technique employed in this study. A
possible explanation for this observation is that the small
amount of phosphate (found in this study to be only 0.3% of
the whole acetone-washed biomass) is not part of the outer
layer (50100 thick). The acetone-washed biomass ma-
terial is quite permeable to lead ions, so that lead may be
bound by inner layers of the acetone-washed yeast biomass
through electrostatic interaction with phosphate.
FTIR-PAS
The photoacoustic IR spectra of an original acetone-washed
biomass preparation and lead-loaded acetone-washed bio-
mass (Fig. 4) are highly complex, reflecting the complex
nature of the acetone-washed biomass composition. In spite
of this complexity several distinctive features typical of ac-
etone-washed biomass components can be observed in the
FTIR spectrum of the native material before its exposure to
lead-containing solution (Nakanishi, 1962; Rao, 1963). A

ASHKENAZY, GOTTLIEB, AND YANNAI: LEAD-BINDING GROUPS IN YEAST BIOMASS 5

 Figure 3. High resolution O 1s XPS spectra of acetone-washed Saccharomyces urarum biomass (a) before and (b) after lead uptake.

broad band in the region 37003000 cm1, with a maximum
at 3290 cm1, is present. This is due to hydroxyl groups that
are hydrogen bonded to various degrees together with the
NH stretching of the secondary amide (RNHCOCH3)
group of the chitin component. Strong absorption of the
carbonyl region at 1657 and 1636 cm1 can be assigned to
the amide I band (CO stretching) of the amide bond in
poly-N-acetylglucosamine (chitin) and the protein peptide
bond present in acetone-washed biomass. These secondary
amide I bands are accompanied by secondary amide II
bands (NH and CN stretching in secondary amides) at
1558, 1540, and 1524 cm1.
A medium strength absorption at 1405 cm1 can be as-
signed to the symmetrical stretching of the carboxylate an-
ion. Strong and complex bands attributed to ether and hy-
droxyl CO stretching appear between 1200 and 940 cm1.
Finally, two weak absorption peaks at 920 and 891 cm1
can be attributed to the glycoside bonds in the polysaccha-
ride structure of the acetone-washed biomass. The absorp-
tion at 920 cm1 probably corresponds to ring vibrations
similar to dioxan (type 1) of b-glycosides. The 891 cm1
band probably corresponds to C1H axial hydrogen bending
vibration in b sugars. The glucan is b-glucan, while the
mannan is a-mannan (Reiss, 1985). This does not necessar-

6 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 55, NO. 1, JULY 5, 1997

Figure 4. Photoacoustic infrared spectra of acetone-washed Saccharomyces urarum biomass before (lower curve) and after (upper curve) lead uptake.

ily imply that the polysaccharide components in the ac-
etone-washed biomass are all b sugars, because according
to Nakanishi (1962) some type 1 bands of the a series
appear in the region of 891 17 cm1.
The only observable change in the spectrum of the lead-
exposed yeast biomass was in the symmetrical stretch of the
carboxylate groups (Nakanishi, 1962), which shifted from
1405 to 1375 cm1. This shift can be attributed to a change
in the counterion associated with the carboxylate anion.
This suggests that acidic groups, especially carboxylate,
contribute to the metallic ion uptake.
ing capacity of the acetone-washed yeast biomass, the spe-
cific biosorption efficiency of each component thought to be
a major metal adsorbing agent was tested. Therefore exam-
ined were the protein fraction, because of its known ten-
dency to interact with heavy metals; chitin, which was
found to be the cell wall component responsible for metal
coordination (Muzzarelli et al., 1980; Tsezos and Volesky,
1982a,b); and mannan, which is the main polysaccharide in
the outer surface of the cell wall (Reiss, 1985).
NaOH-Treated Acetone-Washed Yeast Biomass

Metal Uptake Capacity of Different The role of proteins in lead uptake by S. uvarum was evalu-
Biomass Components ated by examining the metal binding capacity of our ac-
etone-washed yeast biomass preparation with and without
In order to explore the relative contribution of the various
protein. Boiling the preparation with NaOH hydrolyzes the
acetone-washed biomass components to the total lead bind-
proteins and the resulting biomass was washed with water
and acetone.
Table I. Lead uptake from 100-ppm aqueous solution (200 mL) by As a result of this treatment, the number of protein amino
washed yeast biomass components. groups that can be engaged in metallic ion binding is mark-
Metal uptake
edly decreased. Deproteination should, theoretically, de-
capacity Qa crease metal retention. Yet, the opposite was observed
Component (mg/g dry wt) Q/Q0b (Table I). Similar results were reported by Muraleedharan
and Venkobachar (1980), who showed that copper biosorp-
Washed yeast biomass 48.9 1.00
tion by Ganoderma lucidum was more efficient after NaOH
Washed yeast biomass after
8-h boiling with 6% NaOH 56.9 1.16 treatment. The explanation they offered is that the increase
Pure chitin 48.8 1.00 in the metal uptake after the protein removal steps is
Mannan from Saccharomyces uvarum <0.1 0.002 brought about by unmasking of some of the cellular groups,
a
which cannot participate in the sorption process without the
The metal uptake capacity relates to a final lead concentration of 5.25
ppm that represent the lowest residual lead level in all cases except man-
treatment with alkali. Muzzarelli et al. (1980) also found
nan, which did not show an appreciable metal binding capacity. that alkali treatment of Aspergillus niger mycelia improved
b
Q0 is the metal uptake capacity of the washed yeast biomass. their capacity to chelate various metallic ions. Luef and col-

ASHKENAZY, GOTTLIEB, AND YANNAI: LEAD-BINDING GROUPS IN YEAST BIOMASS 7

 Table II. Lead uptake from 100-ppm aqueous solution (200 mL) by 1.5 g of acetone-washed yeast
biomass after chemical modifications.

Control After treatment

Final Metal uptake Final Metal uptake

concn capacity Q0 concn capacity Q
Treatment C0 (ppm) (mg/g dry wt) C (ppm) (mg/g dry wt) Q/Q0a Average

Acetylation 85.4 14.7 81.7 18.9 1.3

43.1 16.1 51.5 23.9 1.5 1.3
39.9 35.4 31.6 40.4 1.1
60.6 34.5 49.4 41.7 1.2
63.3 44.2 15.6 74.0 1.7
63.3 44.2 35.4 55.31 1.3
Succinylation 69.7 26.20 53.3 43.9 1.7
63.8 36.54 50.0 50.1 1.37 1.5
63.8 36.54 52.4 47.83 1.3
TNBS 85.8 7.8 83.8 8.8 1.1 1.1
71.5 25.2 66.3 28.5 1.1
Propylamine 57.2 32.2 92.5 3.4 0.1 0.08
72.4 13.3 98.7 0.73 0.06
a
This expression was calculated on the basis of various final lead concentrations, because the
values shown in the table represent the results for different batches of yeast biomass. It should be
noted that the lead uptake of the control (Q0) was modified by the various treatments, which in turn
resulted in different final lead concentrations after the treatments.

leagues (1991), in trying to explain the increase in zinc
uptake by A. niger after NaOH treatment, suggested that the
removal of certain polysaccharides from the cell wall by
alkali treatment generates more accessible spaces within the
b-glucan chitin skeleton, thus allowing more zinc ions to be
sequestered by this structure. Fourest et al. (1992) treated
mycelial biomass with 0.1 M NaOH before sorption in order
to generate anionic sites without significantly modifying the
cell wall structure. This treatment enhanced the zinc binding
capacity of the cell wall while increasing the equilibrium pH
from 5.0 to 5.9. In the experiments conducted in this study
the equilibrium pH was about 5.7. As was found in previous
experiments carried out in the authors laboratory, the op-
timum pH for lead adsorption was 5.5 and raising it to 9 did
not affect the metal uptake capacity (unpublished data).
Hence, it appears that the interpretation suggested by Four-
est et al. (1992) is not relevant to the investigation reported
here in which lead was used and the biosorbent employed
was produced from yeasts. The improvement in lead uptake
observed in this work after the NaOH treatment was possi-
bly due to better accessibility of the ions to binding moi-
eties. In addition, the N-acetyl moiety of chitin was also
hydrolyzed under the same conditions to the free amino
polysaccharide, chitosan (Onodera and Funabashi, 1991).
Chitosan is known to be superior to chitin in heavy metal
biosorption (Crusberg et al., 1991). Using the Katchalsky
Spitnik equation (Bodek and Kufelnicki, 1995) {pH 4 log
Kh n log [a/(1 a)]}, which correlates the pH with the
degree of neutralization for polymers, it can be estimated,
assuming n 4 1.5 and pKH 4 7.5 for chitosan (Bodek and
Kufelnicki, 1995), that only 5% of the amino groups in
chitosan are charged. From the rest of the amino groups,
some are capable of coordination with metal ions. This may
explain the 16% increase in overall metal uptake capacity
compared to the whole cell wall preparation observed in
this study.
Chitin
Using pure commercial chitin gave a lead binding capacity
of 48.8 mg/g chitin. The acetone-washed biomass metal
uptake capacity was 48.9 mg/g dry wt. Comparison of the
pure chitin with the biomass above was done at the same
final lead concentration (5.25 ppm). Thus, pure chitin has a
lead uptake capacity close to that of the whole acetone-
washed yeast biomass.
The mechanism of biosorption by chitin in the acetone-
washed biomass is probably through coordination to the
unpaired electrons of the nitrogen atoms. Tsezos and Vole-
sky (1982a,b) observed a chitin uptake of 6 mg uranium/g
and 8 mg thorium/g, while whole cells of R. arrhizus ex-
hibited maximum metal uptake of 180 mg U and 170 mg
Th/g dry wt.
Although the respective hypotheses for uranium and tho-
rium uptake mechanisms suggested by Tsezos and Volesky
(1982a,b) are different, in both of them substituted amino
groups of the cell wall chitin network acted initially as
uranium and thorium coordination sites. Coordinated ura-
nium or thorium function subsequently as nucleation sites
for sequestering of additional uranium or thorium.
Therefore, although the pure chitin metal uptake capacity
is very small, chitin may play an important role in the ura-
nium and thorium biosorption by R. arrhizus (Tsezos and
Volesky, 1982a,b).
As can be seen from the experiments with pure chitin
reported here, the uptake capacity for lead is about 6- to

8 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 55, NO. 1, JULY 5, 1997

eightfold greater than that for thorium or uranium reported
by Tsezos and Volesky (1982a,b). However, the chitin con-
tent of the S. uvarum acetone-washed biomass is about 5%
(unpublished data), while the chitin content of the fungus
Rhizopus nigricans, for example, is 58% (Tsezos and Vo-
lesky, 1982a). Muraleedharan and Venkobachar (1990)
showed that a sorbent devoid of chitin removed 89% of the
copper from an aqueous solution, while the whole sorbent
prepared from Ganoderma lucidum removed 94% of the
copper ions. When the concentration of chitin was doubled,
the copper removal capacity was 100%.
Thus, chitin contributed to the metal uptake capacity, but
it did not play a major role in the metal sequestration pro-
cess.
Mannan
As can be seen from the results, the lead uptake capacity of
mannan (isolated by us from S. uvarum) is negligible (i.e.,
it actually was below the detection limit of the analytical
procedure employed in this study). It can be concluded that
mannan does not play a significant role in lead biosorption.
Metal Binding to Biomass after Modification of
Carboxyl and Amino Groups
The metal uptake after the modification of amino and car-
boxyl groups is given in Table II. It can be seen that the
reaction between the carboxyl groups and propylamine sig-
nificantly decreased the metal uptake. The modifications in
the amino groups slightly improved metal uptake. It should
be pointed out that the metal binding efficiency differed
somewhat from one yeast batch to another.
Yet, there was definitely a good correlation between the
results for untreated (control) samples and those for the
same batch after treatment. The fact that the original yeast
samples were of different ages may account for such a vari-
ability.
Blocking the carboxyl groups by treating the biomass
with propylamine brings about a marked decrease in lead
uptake capacity (Table II). Negatively charged carboxyl
groups present in carbohydrates like mannan or glucan,
which occur in the yeast acetone-washed biomass, can ad-
sorb metallic ions by ion exchange. After conversion of the
carboxyl groups into the more hydrophobic amide, an eight-
fold decrease in the metal uptake capacity was observed.
This suggests that the ionic interaction of lead cations with
anionic groups, especially carboxyl groups, is the major
biosorption mechanism responsible for the acetone-washed
biomass metal uptake.
Similarly, Kuyucak and Volesky (1989), who investi-
gated cobalt ions adsorbed by a dead biomass of Ascophyl-
lum nodosum, suggested that the main adsorbing mecha-
nism is through ion exchange. Carboxyl groups in the algi-
nate present in the algal cell wall play an important role.
Crist et al. (1992) concluded that carboxyl groups, which
are present in abundance in uronic acids, could be consid-
ASHKENAZY, GOTTLIEB, AND YANNAI: LEAD-BINDING GROUPS IN YEAST BIOMASS
ered one of the main ligands responsible for the bulk of
metal sorption. However blocking the amino groups by
acetylation or succinylation improved the lead biosorption
(Table II). These experiments were routinely carried out at
pH 5.5, which was found to be the optimal pH for lead and
some other metals sequestration (unpublished results). At
this pH most amino groups in proteins are positively
charged (NH3+). In this state the nitrogen atom cannot co-
ordinate with a cation. Acetylation brings about neutraliza-
tion of the amino groups (Fig. 5). The acetylation reaction
causes an exchange of the positively charged substituted
ammonium ion, which does not contribute to biosorption of
metallic ions, by an amide function (RNHCOCH3) capable
of coordination to the positive metal ion (Tsezos, 1985).
The acetylation reaction decreases the number of posi-
tively charged sites on the acetone-washed biomass surface,
which causes a modest 30% increase in metal uptake ca-
pacity (Table II). The succinylation reaction not only de-
creases the number of positively charged sites in the ac-
etone-washed biomass, but it also increases the number of
negatively charged sites by contributing another carboxyl
group (Fig. 5). This causes a 50% increase in the metal
uptake capacity. The TNBS modification (Fig. 5) has the
same effect as the acetylation reaction because it reduces the
number of positively charged sites on the acetone-washed
biomass. It may be assumed that the TNBS sulfone amide
contribution to the metal sequestering is minor, due to its
size and molecular structure. This modification caused the
same increase of metal uptake as acetylation. This indicates
that the main contribution of the acylation reactions is sim-
ply by reduction of the number of positive sites on the
acetone-washed biomass surface.
The relative contribution of the acylated amines to bio-
sorption by complexation is probably small. Evidence
points at the fact that the main metal sorbing component in
yeast acetone-washed biomass is negatively charged car-
boxyl groups.
CONCLUSIONS
Carboxyl groups were the dominant species in the lead bio-
sorption mechanism by the acetone-washed biomass of the
Figure 5. Substituted amino groups in acetone-washed yeast biomass by
(a) acetic anhydride, (b) succinic anhydride, and (c) trinitrobenzenesulfonic
acid.
9
yeast S. uvarum, but also groups containing nitrogen, such
as in chitin, interacted with lead cations. Such biosorption
mechanisms may also exist in the case of other heavy me-
tallic cations similar to lead.
The authors acknowledge the help of Prof. M. Folman with the
interpretation of the X-ray photoelectron spectroscopy. We are
also grateful to Dr. J. F. McClelland, MTEC Photoacoustics Inc.
(Ames, IA) for conducting the FTIR-PAS measurements.
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