The Role of Chitin in U d u m Adsorption by

R. arrhizus
MARIOS TSEZOS, Department of Chemical Engineering, McMaster
University, Hamilton Ontario, Canada

Summary
In order to further refine and support the uranium biosorption mechanism hypothesis pro-
posed for Rhizopus arrhizus, uranium competitive equilibrium uptake isotherms by chitin were
determined at two different solution pH levels and in the presence of different concentrations of
competing ions, namely, Cu2+, Zn2+, and Fe2+. The co-ion effect became more pronounced as
the co-ion concentration in solution and pH increased. Obtained equilibrium data are in agree-
ment with uranium biosorption data reported earlier. Infrared, mass, and electron paramagnetic
resonance (EPR) spectra of chitin before and after uranium uptake in the presence of the com-
peting ions Cu2+, Zn2+, and Fez+ were recorded. The combination of the spectral data and the
information from equilibrium studies supported the hypothesis advanced earlier on the mecha-
nism of uranium uptake byR.arrkizus. In addition, the data suggested the participation of a free
radical in uranium coordination by the cell wall chitin. The mechanism of reduction of the ura-
nium uptake capacity of the biomass in the presence of competing ions was also elucidated further.

INTRODUCTION

Adsorption is an efficient separation process that, if properly designed, can

selectively and effectively remove adsorbates from a liquid phase into a solid
phase (adsorbent). Furthermore, the separation of the adsorbent, loaded
with the adsorbate from the liquid phase, can be easily accomplished, thus
making adsorption a preferred separation process.
In view of the above advantages of adsorption, a systematic effort has been
undertaken by the author to identify and develop novel efficient adsorbents.
Selected types of inactive waste microbial biomass have recently been identi-
fied as adsorbents that present competitively high radionuclide uptake capac-
ity and selectivity.',* Among them, Rhizopus arrhizus has been shown to pos-
sess very high uranium and thorium uptake capacity. Experimental evidence
available has suggested that specific cellular components of the R. arrhizus
cell wall are responsible for the observed uranium uptake. A detailed ura-
nium uptake (biosorption) mechanism hypothesis has been advanced for the
system uranium-R. arrhizus, according to which the chitin crystalline net-
work in the dead mycellium cell wall acts as the most important cellular com-
ponent in uranium sequestering.3
According to this mechanism hypothesis, amino groups of the cell wall chi-
tin network act initially as uranium coordination sites. Coordinated uranium

Biotechnology and Bioengineering, Vol. XXV, Pp. 2025-2040 (1983)

0 1983 John Wiley & Sons, Inc. CCC 0006-3592/83/082025-16$02.60
2026 TSEZOS

functions subsequently as nucleation site for the adsorption of additional ura-

nium, primarily in the form of hydrated uranyl hydroxide, by the chitin crys-
talline network. The suppression of the biomass overall uranium uptake ca-
pacity that has been observed in the presence of competing ions, such as iron
or zinc, has been interpreted initially as the result of competition of the com-
peting ions for chitin coordination sites and the consequent reduction of the
uranium nucleation sites. The effectiveness of the competing ions in suppress-
ing the overall R.arrhizus uranium uptake has been shown to be a strong
function of solution pH, this has also shown to be the case in the overall ura-
nium uptake capacity of the
In order to obtain additional supportive evidence and to elucidate further the
already advanced uranium biosorption mechanism hypothesis on the subjected
competing ions effect, it was decided to study the behavior of the uranium-
chitin system in detail, in the presence and absence of competing ions and un-
der strictly controlled conditions. The information available in literature on the
uranium uptake of chitin is very limited, largely fragmented and qualitative.
With the above objectives in mind, the present work focused on two main
tasks: the study of the equilibrium competitive uptake of uranium by chitin
and the elucidation of mechanistic aspects of this equilibrium.

MATERIALS AND METHODS

The chitin uranium uptake was determined at pH 2 and pH 4 in the ab-

sence and the presence of three different concentrations of Fe(II), Zn(I1) and
Cu(I1). The above elements were selected because: 1)they are often present in
the waste and process streams of the uranium mining and milling industry
and in waste streams considered as unconventional uranium resources; 2)
they have been shown, under certain conditions, to interfere with the uranium
uptake capacity of R. arrhizus, where chitin has been suggested as the active
Competitive uranium uptake isotherms were determined at 2OoC with
the help of the standard technique used for the determination of activated
carbon adsorption isotherms.
Uranium solutions were prepared by dissolving exact quantities of uranyl
nitrate in distilled, deionized water. Copper, iron, and zinc were introduced
in solution using the same technique and dissolving, respectively, crystals of
CuC12, FeCI2, and ZnClz.
The uranium solution concentration was determined using the Arsenazo
I11 spectrophotometric technique and a Baush & Lomb Spectronic-21 digital-
readout spectrophotometer. The technique was checked against the 235 U de-
layed neutron counting technique, with the help of the McMaster University
nuclear reactor, and was proven more precise partly due to the fact that the
uranyl nitrate used in the experiments was partially depleted in 235U.
Iron, zinc, and copper were determined with the help of a Varian Techtron
AAG atomic absorption spectroscopy unit. ICN Pharmaceuticals unbleached
chitin was used in all experiments with the following elemental composition:
 URANIUM ADSORPTION BY R . ARRHZZUS 2027

C (46.8%), H (6.5%), N (7.30%), and an apparent molecular weight of

770.7.
A significant change in solution pH was observed following contact of the
chitin with the mixed ion solution. Such a change affects the hydrolysis of the
ionic species present in solution, hence their relative composition and adsorp-
tivity. Some form of solution pH control was therefore necessary. Potassium
biphthalate buffer [20% (v/v), 0.05M COOHC6H4COOK], which has been
shown not to interfere with the uranium biosorptive uptake and the uranium
Arsenazo I11 spectrophotometric determination technique, was used for the
pH 4 range.' Furthermore, prior conditioning of chitin to pH 4 was necessary
in order to maintain the solution pH constant at pH 4 throughout each experi-
ment. At pH 2, prior conditioning of chitin alone provided the desired pH
stability. Chitin was conditioned by first washing with distilled water. The
washed chitin was then suspended in water, the suspension pH was adjusted
to the desired level with either NaOH or HCI solutions (BDH reagent), and
allowed to stand overnight. Chitin was then filtered, rinsed, and dried at 65-
70°C. Dry conditioned chitin was ground with a mortar and pestle to finer
particles before use for sorption isotherm determination. The initial solution
uranium concentration was 100 mg/L for all experiments. Concentrations of
100,500, and 1000 mg/L of Fe+2, Zn+2, and Cu2+ were tested for competi-
tive uptake with uranium by chitin. This wide concentration range covers ac-
tual wastewater composition where concentrations up to 1000 mg/L or more
of the competing elements may be encountered. The equilibrium contact time
was 18 h for all experiments and was determined experimentally.
Oxidation of Fe(I1) to Fe(II1) resulted, at pH 4, in the precipitation of ferric
hydrolysis products which interfered with the uranium competitive uptake ex-
periments. In order to avoid macroscopic precipitate (retained by 0.45 pm
filter membrane), air was purged off the distilled water that was used for the
preparation of the uranium-iron contact solution and the contact with chitin
was done in a N2 atmosphere, by bubbling N2 in each of the contact flasks and
subsequently sealing the flask top. Iron-uranium competitive isotherms were
also determined in the dark.
The infrared spectra of the pure and reacted chitin samples were recorded
from KBr disks with the help of a NICOLET FTIR infrared spectrometer, in
the 4000-400 cm-' range of the spectrum. Mass spectra of pure and reacted
chitin were recorded by a VG Micromass 7070 F gas chromatography mass
spectrometer unit. Finally, electron paramagnetic resonance (EPR) spectra
were recorded with the help of a Varian EPR spectrometer.

RESULTS
Uranium Uptake Isotherms
The chitin-uranium uptake was examined in the presence of three different
concentrations of each of the competing ions Cu(II), Zn(II), and Fe(II), and
2028 TSEZOS

at two different solution pH values (pH 2 and pH 4). The resulting uranium
equilibrium uptake isotherms, plots of uranium solid-phase concentration
(chitin uptake q , in mg/g) against the residual equilibrium uranium concen-
tration (Ces,in mg/L), were used to evaluate the effect of the presence of each
of the competing ions on the chitin uranium uptake capacity.
At pH 2, the chitin uptake of uranium (ca. 1 mg/g) was significantly lower
than the uptake of uranium at pH 4 (9 mg/g) under similar experimental
conditions (Fig. 1).At pH 2, the employed analytical technique did not reveal
a discernible reduction in the uranium uptake of chitin, in the presence of
increasing Cu(I1) concentrations (100,500, 1000 mg/L). Similar results were
also obtained in the presence of zinc and iron.
Table I summarizes the observed co-ion effect on the chitin-uranium uptake
at pH 4.It indicates that increasing co-ion concentration from 100to lo00 mg/
L, decreased the observed uranium uptake by chitin by as much as 39%. This
effect was not clear at the 100 mg/L co-ion concentration level but became
better pronounced at lo00 mg/L. Among the three co-ionstested, at pH 4,iron
appeared as the most effective in reducing the chitin-uranium uptake.
Figure 2 presents the chitin competitive uranium uptake isotherms deter-
mined at pH 4 and at four different concentrations of Cu(I1). Similarly, Fig-
ures 3 and 4 present the respective Zn(I1) and Fe(I1) competitive uranium
uptake isotherms.
The presence of potassium biphthalate as a buffering agent in the uranium
solution did not have a discernible effect on the determined competitive ura-

/ *
4 /
9 -
9’
0 - 4 /
/ 4
/
7 - / 4
/
4’

1.- 1 5 -
4’

5E 4 -
d Chili.0
22OC
CoU+6=100mg/I
4 pH-4
0 pH=2

1- 8
0 0 0
1 I I I I I

~ 1 2 3 4 5 6 7 8 9 1 0 1 1

Fig. 1. Chitin-uranium equilibrium uptake isotherms; pure uranium solutions.

 URANIUM ADSORPTION BY R . ARRHIZUS 2029

TABLE I
Co-ion Effect on Chitin Uranium Uptake (9) at pH 4; Percent Reduction of q

100 2 2 3 14 2 6
500 13 12 17 18 11 21
lo00 13 20 19 18 24 39

nium uptake by chitin, as the respective isotherm remained unaffected by the

biphthalate up to 30% (v/v) of the contact solution.
The implemented high concentrations of iron, the ease with which Fe(I1)
oxidizes to Fe(III), and the extensive hydrolysis of Fe(III), even at low pH
values, may result in precipitation of ferric hydroxide in the contact flasks.
Whenever ferric hydroxides precipitated, higher equilibrium uranium con-
centrations were observed, leading to lower calculated uranium uptake ca-
pacities by chitin. Figure 4 illustrates the differences in the observed chitin-
uranium-iron competitive uptake isotherms, at pH 4, and under two
different sets of conditions that, respectively, allowed or prevented ferric hy-
droxides from precipitating. The experimental results reported on Table I
and Figure 4 are free of precipitate (down to 0.45 pm particle size). Experi-

Fig. 2. Chitin-Cu(I1)-uranium competitive equilibrium uptake isotherms.

2030 TSEZOS

Fig. 3. Chitin-Zn(I1)-uranium competitive equilibrium uptake isotherms.

ments at pH 2 were also free of precipitate. The lines depicted on the pre-
sented plots indicate the data trend.

Infrared Spectroscopy
Infrared spectra of chitin samples equilibrated with 100 mg/L U(V1)in the
presence of Fe(II), Cu(II), and Zn(I1) did not reveal any discernible differ-
ences before and after competitive sorption of uranium for all examined
cases.

Mass Spectroscopy
Chitin is not an easily volatilized macromolecule. The degree of decomposi-
tion of the macromolecule was examined over the 25-340°C range by moni-
toring the total intensity of the resulting ionic stream, as a function of the
direct inlet probe temperature. A temperature of 300°C was selected as the
temperature at which the individual mass spectra were subsequently re-
corded. Figure 5 presents a typical pure chitin mass spectrum. Figures 6 and
7 present, respectively, typical mass spectra of chitin equilibrated with ura-
nium alone and with uranium in the presence of Cu(I1) at pH 4. A noticeable
change in the positive ion relative compositions was observed following metal
uptake, indicative of changes occurring on the chitin macromolecule. These
changes are discussed in the subsequent section.
 URANIUM ADSORPTION BY R . ARRHZZUS 203 1

’,/ ’ .
/.
”
0 -
,
*/
- -
8 -
,‘.I *. -0

7 -
,6- /
/

C’
.- .- -.-
., ’0
*,.
1
.

I ::
,D
0
E 4 -
’

0’
.i

.
,

.cD--o
-

0-
- =-
/o/ooo-o---
0

Chitin
22oc
- .-0

P p n =4
3 - Co U+6= 100 mg/I

.
1
Omp/l Fa+?
2 - no pncipitotion I 0 0 mm/I Fo+*
1 0 0 m g / l F.*2
1 - lOOOmg/l Fo+2
~ r o c i ~ i t s t i o n5oome/l
~ ~ F.+Z

1 2
Caq,
3
mop
4 5
U+6 in solution a 10-1
6
-r s 0

Fig. 4. Competitive equilibrium uptake isotherms of the system chitin-uranium-iron, with

and without precipitate formation.

Electron Paramagnetic Resonance Spectroscopy

The paramagnetic nature of iron and copper allowed the recording of EPR
spectra from chitin equilibrated with uranium in the presence of iron or cop-
per. The EPR spectra were also recorded from pure chitin, chitin equilibrated
with 100 mg/L U(V1) alone (pH 4), and chitin equilibrated with 1000 mg/L
Cu(I1) in the absence of potassium biphthalate buffer at pH 4. The pure chi-
tin EPR spectrum exhibited a weak signal at 3365 G (Fig. 8). A weak signal
was also recorded from chitin equilibrated with uranium only (Fig. 9). The
EPR spectra of chitin loaded with uranium in the presence of copper or iron
(Figs. 10 and 11) positively identified both elements as being present on the
chitin macromolecule, along with uranium, in their respective bivalent ionic
states.

DISCUSSION

Effect of Solution p H
The amine nitrogen on each chitin monomer unit has been suggested as the
active site for metal ion coordination. Representing by GN the chitin mono-
mer unit, the dissociation equation of the amine could be represented by:
GN+ : H + H20 + GN: + H30’
2032 TSEZOS

P u r e Chitin

300 'c

Equilibrium is obviously a function of solution pH. Metal ions (including the

uranyl ion or its hydrolysis products) that would coordinate with the lone elec-
tron pair of the amine nitrogen would have to compete with the H30+ ions for
the active sites. A reduced uranium loading on chitin is observed at pH 2, as
opposed to the loading observed at pH 4, most likely due to the increase in

I
I
I
I
I
0.1
.l
I
I
I
I
I
0 1
.I

N
X
I
1
I
Chitin - Uranium ( p H 1 4 )
M I
3W.C

Fig. 6 . Typical mass spectrum of the system chitin-uranium (pH 4).

 URANIUM ADSORPTION BY R. ARRHZZCJS 2033

M/Z

Fig. 7. Typical mass spectrum of the system chitin-uranium-copper (pH 4).

H30+ concentration at the lower pH. Lower chitin loadings at low solution
pH have also been reported in literature by Andreyev et al. who observed a
chitin uranium uptake of 3.5 mg/g at pH 3 and by Muzarelli and Tubertini
who indicated qualitatively a decline in the metal uptake capacity of chitin
and chitosan for an extended series of metals, when the solution pH was re-
duced from 7 to 2.5.4,5

r - "' " " " ' """"""""' """"' " " " " " " " " '"'-

0 +.

Fig. 8. Typical EPR spectrum of pure chitin (pH 4).

2034 TSEZOS

Fig. 9. Typical EPR spectrum of the system chitin-uranium (pH 4).

As reported in the section of Uranium Uptake Isotherms, the competitive

uranium uptake equilibrium data show that chitin exhibits increased selectiv-
ity towards the coordination of the uranyl ion against the co-ions Cu(II),
Zn(II), and Fe(I1) at pH 2. Examining the hydrolysis patterns of the compo-
nents of the system under investigation, we can observe that all three of the
co-ions up to the 1000 mg/L concentration are not expected to produce hy-
drolysis products to any significant extent before the solution pH exceeds 4.6

Fig. 10. Typical EPR spectrum of the system chitin-uranium-copper.

 URANIUM ADSORPTION BY R. ARRHZZUS 2035

' >

-.5 0 +.5

Fig. 11. Typical EPR spectrum of the system chitin-uranium-iron (11).

The uranyl ion, however, hydrolyzes above pH 2.5. It can be calculated that
the approximate proportion of simple UO;', for a lOO-mg/L U(V1) solution,
drops from 100%at pH 2 to 80% at pH 4 and to about 9% at pH 5. Hydroly-
sis species such as U02(OH)+ and (U02)2(0H):+ are common above pH 3.
The other significant change, due to pH increase in the examined system, is
likely to occur through the dissociation of the amine of the chitin macromole-
cule as described above. It appears that. the observed selectivity of chitin for
uranium uptake, at the presence of co-ions at pH 2, is due to the preference of
chitin for the simple H 3 0 + and the uranyl ions that are present in increased
concentrations at low pH.

Effect of Coion Concentration

At pH 4, an increase in co-ion concentration resulted in a decrease in the
observed uranium loading on chitin. This behavior can be interpreted as the
result of the more effective competition of the coions [Cu(II), Zn(II), or
Fe(II)] when their respective concentration in solution is higher. Iron proved
to be more effective than Cu(I1) or Zn(I1) (Table I and Fig. 4). The iron equi-
librium plateau, observed at 1000 mg/L Fe(II), indicates that the uranium
uptake by chitin, under the examined competitive adsorption conditions, is
independent of the solution uranium concentration thus suggesting that the
uranium-iron-chitin system has attained equilibrium. On the basis of the rel-
ative position of the examined co-ions in the Irving-Williams series and con-
trary to the evidence supplied by the experimental results, one would expect
Fe(I1) to be less effective than Cu(I1) and Zn(I1) in competing for coordina-
tion. Spectral examination of the adsorption system has, however, indicated
that the observed deviation may be an apparent one, as the ferrous ion ap-
2036 TSEZOS

pears to interfere with the uranium uptake by chitin (at pH 4) not only
through competition for the available coordination sites, but also through the
deposition of coloidal iron hydrolysis products on the chitin particles. As a
result, the effect of ionic ferrous ion on the chitin uranium uptake, through
competition as a Lewis acid alone may be below that of Cu(I1) or Zn(I1) as
predicted by the Irving-Williams series. The co-ion uptake by chitin in the
presence of uranium was very low so that an accurate estimate through the
determination of the difference in co-ion concentration in solution, following
adsorption, was not possible.

Statistical Significance of Equilibrium Experimental Results

Uranium equilibrium solution concentration was determined spectropho-
tometrically. The precision of the determined uranium equilibrium concen-
tration values, at a 95% confidence interval and at 50 mg/L uranium concen-
tration, was determined to be better than f 3 % , regardless of the presence or
absence of coions.
A similar analysis using the delayed neutron counting technique for
Uranium-235 indicated a 60% depletion of Uranium-235 and a 95% confi-
dence interval of f 11 and f 9%, respectively, at 30 and 60 mg/L Uranium-
235. The 95% confidence interval, calculated for the overall procedure of
determining uranium loading on chitin, was evaluated at q = 8.8 mg/g
as f 4%.

Infrared Spectroscopy
This technique was not sensitive enough to provide additional information
on the nature of interaction among chitin and the co-ions in solution. The
recorded pure chitin infrared spectrum corresponded well with the chitin
spectra reported in l i t e r a t ~ r e The
. ~ effect of metal coordination on the fre-
quency of the Amide I (1660 cm-') and I1 bands (1565 cm-l) was not ob-
served as expected. Shifts in these bands have, however, been reported in the
literature, but for systems other than the ones examined.8 It can be calculated
that the observed uranium uptake of 9 mg/g corresponds to the coordination
of approximately 1 uranyl group per 130 chitin monomeric units, a rather
limited ratio that may justify the observed results.

Mass Spectroscopy
Mass spectral studies were undertaken in order to provide further evidence
on the site of metal uptake by chitin. A temperature of 300°C was chosen for
recording the reported mass spectra because at that temperature a more sta-
ble and rich fragmentation pattern was obtained. Table I1 presents a list of
fragments that can be attributed to the ions detected in the recorded mass
spectra. Similar assignments have been reported in the l i t e r a t ~ r eTable
.~ I11
summarizes the peak ratios that were used to characterize the interaction be-
tween chitin and the metals in solution.
 URANIUM ADSORPTION BY R . ARRHIZUS 2037

TABLE I1
Mass Ions and Associated Ion Fragments

Fragment
~~

28 )C=O
31 -CH,OH
0
It
43 -C-CH,
I
55 -CHCHNH,C
I
0
II
58 -NHCCH3
0
II
71 CHNHCCH,
I
72 -COHHCHN,C
I
0
II I
84 -CHNHCCH$H
I

Following the coordination of a metal ion to the chitin amine nitrogen, it is

expected that fragments associated with the amino group will reduce their
contribution to the total ionic current I . Fragments from the acetylated amino
groups could be those at m/z = 84, 71, and 58, and from the deacetylated
amino groups at m/z = 55. One may also assume, on the basis of the pro-
posed mechanism, that the contribution of the -CH20H group (m/z=31) to
the total ionic current should be independent of the coordination of a metal
ion to the chitin nitrogen, which lies diagonally and on the other side of the
molecular plane. Consequently, following a Lewis acid-base reaction of chi-
tin with metal ions, as the contributions of the fragments from the acetylated
and deacetylated amino groups are reduced, the ratios of the respective peaks

TABLE 111
Peak Ratios in Mass Spectra from Pure Chitin and Metal-Bearing Chitin (pH 4)

Sample
Ratio Chitin Chitin-U Chitin-U-Cu Chitin-Fe Chitin-U-Fe Chitin-U-Zn

55/31 17.5 6.3 5.1 5.1 8.1 10.2

71/31 7.5 3.2 2.4 3.0 4.5 5.2
84/31 3.6 1.4 1.1 1.0 1.6 2.0
58/31 0.9 0.5 0.4 0.6 0.7 0.6
2038 TSEZOS

to the -CH20H ( m / z = 31) peak should also decrease. The data on Table I11
confirm this behavior not only for the uranium chitin system but also for a
variety of systems of chitin with complexing metal ions, under competitive
and noncompetitive uptake conditions. These results provide indirect sup-
portive evidence for the hypothesis that the chitin nitrogen is the principal
uranium coordination site on the chitin macromolecule.

Electron Paramagnetic Resonance (EPR) Spectroscopy

The pure chitin EPR spectrum exhibited a weak peak at 3365 G (Fig. 6).
Such a singlet has been observed and reported in literaturelo as typical of
chitin and chitosan. The signal can be attributed to an unpaired electron
from a free radical on the chitin macromolecule, tentatively a hydroxyl group.
Following the uptake of uranyl ions (at pH 4), the EPR spectrum of pure
chitin showed a splitting. This apparent threefold splitting of the uranium-
chitin spectrum is suggested as due to the unpaired electron density on the
nitrogen atom (I= 1, 21+ 1=3), which in turn is complexed with a uranyl
ion. This unpaired electron may originate from the chitin free radical that was
detected in the pure chitin spectrum, with the charge density redistributed
following uranium complexation with the amine nitrogen.
The EPR spectrum of chitin-copper (11) complex was recorded in the ab-
sence of potassium biphthalate buffer (an oxygen bearing ligand) at pH 4.
The recorded spectrum corresponded well with the spectrum reported by Hall
and Yalpani for a similar system." However, part of the nitrogen hyperfine
structure of the present spectrum was better resolved and suggests a Cu2+-
nitrogen complex.
An ionic copper (11)-chitin complex was also detected following uptake of
uranium by chitin in the presence of Cu(I1) (pH 4) (Fig. 10). The relatively
large anisotropy of the g parallel component suggests that the ligands are co-
ordinating via at least two oxygen atoms. The smaller size of the perpendicu-
lar copper nuclear hyperfine splittings indicates that the unpaired electron
resides in the d copper orbital, hence pointing to an ionic Cu2+ complex.
Finally, the partially unresolved structure shown on the perpendicular com-
ponents suggest the presence of nitrogen as one of the coordinating ligands.
This structure was better resolved in the simpler chitin-copper system. De-
tailed computer simulation of the chitin-coppei system EPR spectrum also
suggested that at least one nitrogen is involved in the chitin-metal coordina-
tion, and an axially symmetrical copper (11)complex with chitin, through an
amine nitrogen has been suggested.12The simultaneous presence on chitin of
ionic copper (11) with coordinated uranium (as indicated by the observed ura-
nium uptake) supports the hypothesis that copper present in solution as a
coion with uranium, can compete for nitrogen coordination sites and can
result in a reduction of the overall uranium equilibrium uptake of chitin.
Similarly the EPR spectrum of chitin equilibrated with 100 mg/L U(V1) in
the presence of 1000 mg/L Fe(I1) at pH 4, indicates (Fig. ll),through the two
sharp signals at 1600 and 3360 G, the presence of ionic ferrous ion on the
 URANIUM ADSORPTION BY R . ARRHIZUS 2039

chitin matrix. This ionic Fe(II), as similarly the case with Cu(II), can be sug-
gested as the co-ion which through competition results in a reduction of the
observed overall uranium uptake. In addition, the broad signal observed on
the spectrum (1000-4000 G ) is indicative of the presence of colloidal iron in
the chitin crystalline network. Ferrous ion hydrolyzes and oxidizes easily to
Fe(III), which, in turn, yields solutions supersaturated to the solid phase,
usually FeO(OH).6 It can be suggested that the reduced solubility of the iron
hydrolysis products increases their absorptivity, hence resulting in adsorption
of hydrolyzed Fe(II1) by the chitin particles. Adsorbed ferric ion hydrolysis
species, in turn, result in a further reduction in the observed uranium uptake
by preventing uranyl ions from having access to chitin coordination sites. The
above suggested dual mechanism of interference of iron with uranium uptake
by chitin may account for the apparent reversal in the Irving-Williams series,
observed at pH 4.

MECHANISTIC CONSIDERATIONS FOR R.Arrhizus

The data acquired thus far provide further evidence that uranium forms a
complex with the chitin nitrogen. A free radical, tentatively assigned to one of
the chitin hydroxyl groups, is assumed to be participating in the complex for-
mation. This is feasible due to the crystalline nature of chitin which consists
of stacked chains of acetylated and non glucosamine units linked by oxygen
bridges.8 The free radical may belong to a monomer unit adjacent to the unit
which has formed the complex with the uranyl ion.
The importance of the limited uranium, taken up by chitin through coordi-
nation (process A), as nucleation sites for further adsorption of radium by the
mycellium cell wall, was shown again. The results indicated that, similarly to
the behavior observed for the whole inactive mycellial cells, chitin uranium
uptake is reduced at pH 4 in the presence of co-ions. This is affected by the
competition of the co-ions for the nitrogen site of the chitin crystalline net-
work. The competition reduces the nucleation sites generated through ura-
nium coordination to the chitin network in the mycellial cell wall (process A)
resulting in to the observed reduction in the overall biosorptive uranium up-
take of R.arrhi~us.~ In addition, the higher selectivity of chitin for uranium,
at pH 2, along with the reduced chitin uranium uptake at this pH level are
also in accordance with the uranium uptake behavior of the inactive R.arrhi-
zus cells. Furthermore, the suggested steric hinderance of uranium, caused
by the deposition of iron oxidation-hydrolysis products on the external surface
of chitin, can in a similar manner result in the experimentallyobserved decrease
of the overall uranium biosorptive capacity of R.arrhizus, over the decrease
that should be expected through competition for complexation alone.
In terms of process application, the selectivityof biosorption for uranium is
increased at pH 2, while competition by co-ions in solution not only depends
on the strength of their complex formation ability but also on their hydrolysis
pattern at higher solution pH values.
2040 TSEZOS

CONCLUSIONS

Uptake of uranium by chitin is indicated as occurring by coordinating ura-

nium to the chitin nitrogen according to the I-wing-Williams series. This is
supported by new spectral evidence. Hydrogen ions are particularly effective
in competing with uranyl and other metal ions. A free radical, indicated as
present on the chitin macromolecule and tentatively assigned to a hydroxyl
group, appears to participate in the uranyl ion coordination to nitrogen.
Co-ions that hydrolyze extensively may reduce the uranium uptake capac-
ity of chitin by competing for nitrogen coordination sites, and by physically
blocking uranium from having access to active coordination sites through the
deposition of colloidal hydrolysis species on the chitin particles. These effects
are transferred to uranium adsorption by R.arrhizus through process A of the
proposed mechanism.
The uranium biosorption mechanism hypothesis advanced earlier by the
author is supported by the present work and is elucidated further by refining
the understanding of the effect of coions on uranium uptake by the inactive
biomass and by indicating the participation of a free radical in the uranium
nitrogen bond.

The author wishes to acknowledge the assistance of Ms. A. M. MacNamara in executing the
experimental work. Thanks are also due to Dr. S. Mattar (Department of Chemistry, University
of Toronto) for his valuable assistance in recording and interpreting the EPR spectra. The
present work has been supported through a grant from the National Sciences and Engineering
Research Council of Canada.

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Accepted for Publication February 8, 1983