Biosorption of Uranium by Pseudomonas
aeruginosa Strain CSU: Characterizat ion
and Comparison Studies
Michael 2.-C. Hu, John M . Norman, Brendlyn D. Faison,
and Mark E. Reeves*
Chemical Technology Division, Oak Ridge National Laboratory, Oak Ridge,
Tennessee 37831-6194, USA; e-mail: mri@ornl.gov
Received August 21, 1995/Accepted January 25, 1996
Pseudomonas aeruginosa strain CSU, a nongenetically
engineered bacterial strain known to bind dissolved hexa-
valent uranium (as UOf+and/or its cationic hydroxo com-
plexes), was characterized with respect to its sorptive
activity (equilibrium and dynamics). Living, heat-killed,
permeabilized, and unreconstituted lyophilized cells were
all capable of binding uranium. The uranium biosorption
equilibrium could be described by the Langmuir iso-
therm. The rate of uranium adsorption increased follow-
ing permeabilization of the outer and/or cytoplasmic
membrane by organic solvents such as acetone. P. aeru-
ginosa CSU biomass was significantly more sorptive to-
ward uranium than certain novel, patented biosorbents
derived from algal or fungal biomass sources. P. aerugi-
nosa CSU biomass was also competitive with commercial
cation-exchange resins, particularly in the presence of
dissolved transition metals. Uranium binding by P. aeru-
ginosa CSU was clearly pH dependent. Uranium loading
capacity increased with increasing pH under acidic condi-
tions, presumably as a function of uranium speciation
and d u e to the H' competition at some binding sites.
Nevertheless, preliminary evidence suggests that this mi-
croorganism is also capable of binding anionic hexava-
lent uranium complexes. Ferric iron was a strong inhibitor
of uranium binding to P. aeruginosa CSU biomass, and
the presence of uranium also decreased the Fe3+loading
when the biomass was not saturated with Fe3+,suggest-
ing that Fe3+and uranium may share the same binding
sites on biomass. Although the equilibrium loading ca-
pacity of uranium was greater than that of Fe3+,this bio-
mass showed preference of binding Fe3+over uranium.
Thus, a two-stage process in which iron and uranium are
removed in consecutive steps was proposed for efficient
use of the biomass as a biosorbent in uranium removal
from mine wastewater, especially acidic leachates. Q 1996
John Wiley & Sons, Inc.
Key words: biosorption sorption uranium iron Pseu-
domonas aeruginosa bacteria remediation
INTRODUCTION
Application of biosorption technology to the treatment
of radionuclide-containing, or nuclear, waste streams
has been given significant attention recently by the re-
search Uranium is one of the most seri-
ous contamination concerns because of its radioactivity
and heavy-metal toxicity. Uranium biosorption by vari-
* To whom all correspondence should be addressed.
Biotechnology and Bioengineering, Vol. 51, Pp. 237-247 (1996)
0 1996 John Wiley & Sons, Inc.
ous microorganisms and related biopolymers has been
reported and summarized repeatedly.6-s,20-22Dat a from
the referenced studies document that biomass from fil-
amentous fungi such as Aspergillus niger, Rhizopus ory-
zae, and Penicillium sp.; yeasts such as Saccharomyces
cerevisiae; algae such as Chlorella regularis; actinomy-
cetes such as Streptomyces longwoodensis and S. virido-
chromogenes; and unicellular bacteria such as Citro-
bacter sp., Zoogloea ramigera, and Pseudomonas
aeruginosa, are capable of uptake or binding of uranium
greater than 15% of biomass, dry weight. A metal load-
ing capacity of greater than 15% of biomass (dry weight)
has been defined as an economic threshold for practical
applications of biosorption when compared with alter-
native methods such as traditional adsorption, ion ex-
change, chemical precipitation, solvent extraction, and
reverse o s r n o ~ i sIt. ~should
~ ~ ~ be noted that the values
and comparisons reported in the literature for uranium
loading capacity only have a relative meaning because
of different testing conditions (e.g., temperature, pH,
and wastewater composition) and methods. In addition,
uranium biosorption mechanisms by biosorbents of vari-
ous biological origins may be different. Processes such as
complexation, ion exchange, coordination, adsorption,
chelation, and microprecipitation may be synergistically
or independently involved in the metal b i o ~ o r p t i o n . ~ ~
The current work was undertaken as part of a technol-
ogy development effort directed at restoration of ura-
nium mining and milling sites, including locations where
acid mine leachates are a particular problem. As a first
step in the biosorption technology development, various
microorganisms were screened for uranium sorption ca-
pacity. As discussed later, P. aeruginosa strain CSU,
which was isolated by Johnson et al.I7 at Colorado State
University, and originally identified as useful for ura-
nium removal at Oak Ridge National was
selected as the best candidate for studies of uranium
bisorption performance characterization. Both chemical
and physical pretreatments were tested as a means of
enhancing the biomass loading capacity. Results ob-
tained by using freeze-dried cells of P. aeruginosa CSU
were compared with those obtained by using biosor-
bents developed by other research groups, commercial
CCC 0006-3592/96/020237-11
ion-exchange or chelation resins, and inorganic hydrous
titanium phosphate resins prepared by the sol-gel tech-
nique. Major factors affecting uranium biosorption by
P. aeruginosa CSU biomass, such as pH, temperature,
and interference by other cations and anions, were
also investigated.
MATERIALS AND METHODS
Preparation of Chemicals
and Experimental Solutions
Uranium in nitrate salt form, UO2(NO3)2. 6H20 (ana-
lytical grade), was purchasedfrom J. T. Baker Chemical
Co. (Phillipsburg, NJ). Stock solutions containing ap-
proximately 10,000 mg of uranium per liter and diluted
solutions therefrom were prepared at room temperature
in distilled and deionized water (ddH20) from a Milli-
QTM Water System (Millipore Laboratories, Bedford,
MA). This resulted in an acidic solution (pH ca. 2.5)
with all the uranium salt in solution with no obvious
precipitates. All prepared culture media, such as yeast
malt broth, potato dextrose broth, and marine broth,
were purchased from Difco Laboratories (Detroit, MI).
The sodium nitrate, potassium nitrate, ferric sulfate, and
ferric nitrate were of analytical reagent grade and were
obtained either from Sigma Chemical Co. (St. Louis,
MO) or Aldrich Chemical Co. (Milwaukee, WI). Or-
ganic solvents, such as HPLC reagent-grade acetone,
ethanol, and methanol, were supplied by EM Science
(Cherry Hill, NJ).
All glassware for the biosorption experiments was
routinely washed with 1.ON HN03 and rinsed exten-
sively with ddH20 to prevent interference by contami-
nants. The walls of polypropylene or Nalgene plastic
containers, often used in our experiments, did not bind
uranium and other metals from solution. The pH of
each solution was measured by a digital pH meter and
adjusted by the addition of 1.ON HN03 or 1.ON NaOH
except where otherwise described. An aqueous analyti-
cal standard solution containing loo0 mg/L uranium in
2% HN03 was purchased from Spex Industries, Inc.
(Edison, NJ).
Maintenance and Cultivation of Microorganisms
Cultures of Aspergillus niger ATCC 9642, Citrobacter
freundii ATCC 8454, Saccharomyces cerevisiae NRRL
Y-2574, and Streptomyces longwoodensis ATCC 29251
were maintained on YM agar. Rhizopus arrhizus ATCC
58106 and Rhizopus oligosporus ATCC 22959 were
maintained on potato dextrose agar. All these stock
cultures were incubated and stored at ambient tempera-
ture (22-25°C). Pseudomonas atlantica was maintained
on marine broth agar. Azofobacter vinelandii was main-
tained on Burke’s media agar. Mixed cultures (environ-
238 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 51, NO. 2, JULY 20, 1996
mental isolates) in this work were grown anerobically
in freshwater enrichment medium and incubated at am-
bient temperature with agitation (125 rpm) on a New
Brunswick Model G2 Gyrotory Shaker for 14 days. Fol-
lowing enrichment, the cultures were transferred to nu-
trient broth. Experimental cultures were inoculated into
the corresponding liquid broth medium (nutrient broth
for the environmental isolates) and incubated at ambi-
ent temperature. Cultures of unicellular bacteria and the
yeast were incubated until the midexponential growth
phase. Filamentous fungi and bacteria were incubated
until mycelial pellets approximately 5 mm in diameter
were produced. For harvesting, the cultures of nonfila-
mentous organisms were centrifuged at 12,OOOg in a
Sorvall RC-5B centrifuge at 26°C for 20 min. Filamen-
tous cultures were harvested by suction filtration
through a 0.45-pm filter and homogenized for 10 min
in a blender. The microbial biomass (pellets or filter
cakes) was washed three times with ddH20.
P. aeruginosa CSU was obtained from the laboratory
stocks. The composition of M9 medium for growth of
P. aeruginosa CSU contains (per liter): 6.8 g Na2HP04
(anhydrous); 3.0 g KH2P04;0.5 g NaC1; and 1.0 g NH4C1.
After sterilization of this medium in a autoclave at 121°C
(15 psi) for 20 to 30 min, the following filter (0.45-pm)-
sterilized aqueous solution was added: 2 mL of 1 M
MgS04; 0.1 mL of 1 M CaC1,; and 10.0 mL of 40%
(v/v) sodium lactate. P. aeruginosa CSU was routinely
maintained on a 1.5 wt% Bacto-Agar slant containing
M9 medium and stored in a refrigerator at 4°C. Every
2 to 4 weeks, the CSU cells were transferred from the
old slant to a fresh slant. For cell cultivation, the CSU
cells were transferred (with a loop) from the stored slant
to a fresh slant, which was incubated in an incubator at
30°C for 1 to 2 days. Twenty milliliters of M9 liquid
medium in a 100-mL Erlenmeyer flask was inoculated
(with a loop) by the cells growing on the incubated agar
slant and incubated on a rotary shaker at 200 rpm and
30°C for 24 to 30 h. Next, 5 mL of the growing cell
culture in the flask was aseptically transferred to a 2-L
volume of M9 medium in a 3-L Erlenmeyer flask, which
was incubated on a rotary shaker at 200 rpm and 30°Cfor
24 to 30 h. This 2-L growing culture was then aseptically
transferred to 300 L of sterilized M9 medium (with
AntiFoam B Silicone Emulsion, 15% to 17%) in a
500-L New Brunswick Model IF-500 fermentor (New
Brunswick Scientific Co.). After approximately 30 h of
fermentation (culture OD660nm + O X ) , the CSU cells
were collected by passing the culture through a Sharp-
lesTMtype AS26NF continuous spinning-bowl centri-
fuge system (Pennsalt Chemicals Corp., Philadelphia,
PA). The paste was resuspended and washed with
ddH20, centrifuged, and lyophilized with a Labconco
Lyph Lock 4.5 freeze-dry system (Labconco Corp. Kan-
sas City, MO). The biomass production yield was ap-
proximately 0.5 g of lyophilized biomass per liter of
growth medium. The biomass was stored in a freezer
(- 15°C) until use.
Comparison with Biosorbents and Ion-Exchange/
Chelating Resins
Some fungus- or alga-based biosorbents (designated as
VS#1 through VS#8; for details, see Table IV) were
kindly supplied by Dr. B. Volesky at McGill University,
Canada. CompozymeTMwas obtained from Mekong
Market, Inc. (Columbus, OH). NovosorbTMwas ac-
quired from Novo Laboratories (Danbury, CT). All
organic-matrix-based resins were purchased from com-
mercial companies: Bio-Rad Laboratories (Richmond,
CA); Rohm and Haas (Philadelphia, PA); Supelco, Inc.
(Bellefonte, PA); Dow Chemical Co. (Midland, MI); or
J. T. Baker Chemical Co. (Phillipsburg, NJ). They were
used as supplied, without pretreatment. Two types of
inorganic-matrix-based resins, which have been success-
fully developed at Oak Ridge National Laboratory
(ORNL) for the treatment of radioactive wastewater,
were kindly supplied by Dr. J. L. Collins.
Analyses of Uranium and Other Metals
Dissolved uranium in solution was determined either
by the Arsenazo I11 method27or by inductively coupled
plasma atomic emission spectrometry (ICP-AES)
(Perkin-Elmer Plasma 400, Perkin-Elmer Cop., Nor-
walk, CT). In Arsenazo I11 methods, uranium can be
measured colorimetrically after reaction with Arsenazo
I11 (Aldrich). This assay, which measures uranium accu-
rately over a 0.54- to 60-ppm range, was modified when
necessary by diluting the Arsenazo I11 reagent by a
factor of 4, allowing accurate measurement in the range
of 0.02 to 10 ppm uranium. Spectrophotometric mea-
surements at 650 nm were performed on a Varian Cary
Model 219 instrument (Varian Associates, Inc., Palo
Alto, CA). The ICP analyses for dissolved uranium and
ferric iron were made at 409.014 and 238.863 nm, respec-
tively. Experimental samples were centrifuged or fil-
tered through a 0.45-pm membrane syringe filter before
being introduced into the ICP system. Tests were con-
ducted to ensure that the filter apparatus did not adsorb
dissolved uranium (data not shown).
Uranium Sorption Experiments
Except as otherwise described, for all uranium-binding
equilibrium experiments and parallel tests, CSU bio-
mass (30 to 50 mg, dry weight), biosorbents (50 mg), or
resins (100 mg) were contacted with 30 mL of uranyl
nitrate solution (pH adjusted to 2.4 by HN03) in a
50-mL polypropylene centrifuge tube that was shaken
on an orbital shaker at 200 rpm and 22°C. Contact time
was approximately 24 h. Uranyl nitrate solutions con-
taining 0 to 1000ppm uranium were prepared for deter-
HU ET AL.: BIOSORPTION OF URANIUM BY P. AERUGlNOSA
mination of sorption isotherms. The low pH ensured
that all the uranium (U) was soluble at these concentra-
tions, and experimental controls verified that noted re-
ductions in U concentrations were due to biosorption
and not to precipitation. The metal absorbed by sor-
bents or resins (4,mg metal/g dry sorbent) was calcu-
lated as:
where Co is the initial metal concentration (mg/L), Cfis
the final metal concentration after contact with sorbent
(mg/L), V is the solution volume (mL), and W is the
sorbent weight in dry form (mg). In this work, metal
removal percentage, UR % (= 100(Co - Cf)/Co) was
also used as a comparison parameter.
In parallel testing, the first set of experiments was
performed using a pure uranyl nitrate solution as de-
scribed above. Subsequent experiments were designed
to study the utility of these sorbents for uranium re-
moval in the presence of interfering cations such as
Fe3+,A13+,Ca2+,and Mg2+,as are usually found in real-
world wastewater.
For uranium biosorption dynamics studies, 500 mg
of dry P. aeruginosa CSU biomass was contacted with
500 mL of an approximately 100-ppm uranium solution
agitated vigorously by a magnetic stirrer. Samples
of the homogeneous suspension were withdrawn by a
10-mL disposable syringe at a predetermined time and
forced through a 0.45-pm membrane microfilter to pre-
vent further uranium sorption by the biomass. Tests
were conducted to ensure that the filter apparatus did
not absorb dissolved uranium (data not shown).
The effects of operating conditions such as pH and
temperature on the uranium sorption were also studied.
Aqueous solutions of nitric acid, sulfuric acid, and so-
dium hydroxide were used to adjust the solution pH. An
ice-water bath was used to maintain low temperature.
Effect of Biomass Pretreatments
on Uranium Sorption
In an effort to study the effects of various pretreatment
procedures, portions of the CSU cell slurry or biomass
powder were resuspended in various reagents [1.0 g of
cells (dry weight) per 10 mL of reagent]: (1) inorganic
solutions; (2) organic solvents; or (3) surfactants and
enzymes. Heat treatment was also used. After each pre-
treatment, the CSU was tested for uranium uptake ca-
pacity.
Interference of Cations and Anions
with Uranium Sorption
The CSU cells were contacted for 2 h with solutions
containing 95 mg/L uranium [equivalent to 0.4 mmol
U02(N03)]2 plus 0.4 mmol of the interfering cation,
239
also as its nitrate salt. The control received a total of
0.80 mmol of U02(N0&. The final cell concentration
was 1.0 g dry weight/L, and the total volume was
50 mL. Various uranium/ferric iron ratio mixtures
(pH 2.5) were prepared from uranyl nitrate and ferric
nitrate stock solutions for further study of Fe3+inhibi-
tion of uranium biosorption. In each case, 40 mg CSU
biomass was used to contact 30 mL of mixture, which
was contained in a 50-mLpolypropylene centrifuge tube
and shaken on an orbital shaker at 250 rpm and 22°C
for 20 h. After centrifugation (10,000 rpm for 10 min),
a 10-mL volume of sample was withdrawn and passed
through a 0.45-pm microfilter for ICP analysis of ura-
nium and iron. Mixtures of a series of sulfate concentra-
tions (0,100,1000,5000,10,000,20,000,and 30,000 ppm)
from Na2S04and 100 pprn in uranium were prepared
to determine the effect of SO:- on uranium sorption by
P. aeruginosa CSU. Meanwhile, mixtures of equivalent
sodium concentrations, as in the above mixtures but
from NaN03, were prepared as controls.
RESULTS
Screening of Microbial Organisms
for Uranium Sorption
An exhaustive survey and assessment of the microbio-
logical literature on uranium biosorption (i.e., the bind-
ing of uranium to biological tissues incorporating ad-
sorption, ion exchange, chelation, transformation, etc.)
led to the identification of six organisms of potential
utility. As used in an earlier work by Horikoshi et a1.,16
the major criterion for the identification was based on
the reported values of maximum uranium uptake capac-
ity (weight percentage of bound uranium over dry bio-
sorbent). These six organisms are Aspergillus niger
ATCC 9642 (a filamentous fungus, deuter~mycete)?~
Pseudomonas aeruginosa CSU (a unicellular bacterium,
Gram-negative),26 Rhizopus arrhizus ATCC 58106
(a filamentous fungus, zygomycete)?2 Rhizopus
oligosporus ATCC 22959 (a filamentous fungus, zygo-
mycete)?l Saccharomyces cerevisiae NRRL Y-2574
(a yeast fungus, ascomycete),26 and Streptomyces
longwoodensis ATCC 29251 (a filamentous bacterium,
Gram-positive).' In addition to the organisms listed
above, we also included Citrobacter freundii ATCC 8454
(a unicellular bacterium, Gram-negative) from our labo-
ratory's culture collection in the testing regime. This
organism had shown promise as a biosorbent material
for uranium in earlier testing in our laboratory (data
not shown).
Enrichment methods were used for isolating organ-
isms with uranium-binding ability from environmental
samples. Mixed cultures (environmental isolates) were
obtained by sampling soil adjacent to, and sediment
contained within, a small streambed in Rowan County,
240 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 51, NO. 2, JULY 20, 1996
NC. The mixed culture from soil contained bacteria with
one to three morphological types, all of which were
unicellular, predominantly Gram-negative, and only a
few motile species. The mixed culture from sediment
contained bacteria with greater than three morphologi-
cal types, all of which were unicellular, predominantly
Gram-negative, and some motile species.
A total of 11 biosorbent candidates, including seven
commercially available axenic cultures, two mixed cul-
tures obtained by environmental sampling and enrich-
ment, and two proprietary commercial biosorbent prep-
arations (CompozymeTM and NovosorbTM), were
chosen for preliminary screening. Since most of the re-
ports in the literature described uranium sorption exper-
iments carried out under widely varying conditions of
uranium concentration, temperature, and pH, prelimi-
nary screening consisted of a test performed under stan-
dard conditions. These biosorbents were exposed to un-
buffered uranium (VI) solution (uranyl nitrate in
ddH20,95 ppm uranium initial concentration, pH 3.5) at
room temperature. Buffer was not used so that possible
interference by buffering components was eliminated.
Uranium bisorption was determined indirectly by assay
of dissolved uranium remaining in solution after reac-
tion with Arsenazo I11 reagent. P. aeruginosa CSU and
R. arrhizus exhibited the most rapid and most complete
removal under these experimental conditions (Fig. 1).
The fast dynamic behavior of uranium removal suggests
a minimal residence time (or contact time between
wastewater and sorbent) for a process based on this
organism. Subsequent screening tests were carried out
among siderophore-producing bacteria. P. aeruginosa
CSU, Pseudomonas atlantica, and Azotobacter vinelan-
dii (Fig. 2). These three bacteria also give good produc-
tion of extracellular biopolymer (i.e., polysaccharide),
which may be involved with metal binding. P. aeruginosa
B
-90
,p 80
5 70
2
S 60
.e 50
M
.-
.z
40
2 30
(Y
20
$
.- 10
9
g o
o 4 8 12 16 20 24 28
Contact time (h)
Figure 1. Preliminary screening of biosorbent for uranium removal.
( 0 )R. urrhizus; ( 0 )P. ueruginosa; (+) A. niger; (V)Mixed culture
from sediment; (IMixed
) culture from soil; (V)S. longwoodensis;
(A) S. cerevisiue; (0) R. oligosporus; (0) Compozymem; (A) C.
freundii; (W) Novosorbm.
 I6O
3 160
5ble exception was hydrogen peroxide, an oxidizing
agent, which may have increased uranium removal and,
hence, loading slightly, However, oxidizing acids did
.O 140
P not show an analogous effect when compared with a
nonoxidizing acid. Alkaline pretreatment inhibited ura-
nium binding to a marked degree when NaOH concen-
tration was higher than 50 mN (data not shown). Pre-
treatment of cells with organic solvents significantly
increased uranium removal and loading (Table I). This
effect appeared to parallel solvent polarity. Acetone,
the most polar solvent in these tests, resulted in a more
than twofold increase in uranium binding.

: ;
00 200 400 600 600 1000 1200
1400
Uranium in liquid (mg/L)
Figure 2. Uranium biosorption by microbial biomass from sidero-
phore-producing organisms. Conditions: 22°C; pH 2.4; overnight con-
tact. (0)P. aeruginosa CSU; (0) P. atluntica; (A) A. vinelandii.
CSU showed the highest affinity (at low uranium con-
centrations) and maximal capacity (approximately
100 mg U/g, dry weight, at pH 2.4) and, thus, was identi-
fied as the lead candidate for further study.
Effect of Pretreatments on Uranium Biosorption
by P. aeruginosa CSU
Autoclaving and boiling of the CSU cells caused some
loss of uranium-binding capability (Table I). However,
oven heat did not harm the uranium binding and, in
some cases, even enhanced it by approximately 50%
relative to the untreated CSU cells. Pretreatment of
CSU cells with various inorganic reagents (<50 mN)
had little effect (Table I). More acidic pretreatments
(50 mN) caused a decrease in uranium binding, which
was particularly significant in the case of H2S04.A possi-
Heat-killed lyophilized CSU biomass (autoclaved at
121"C, 15 min), living cells in the absence of organic
nutrients, and living cells preincubated with 1%NaN3-
bound uranium essentially as well as did living cells in
a 2-h contact (data not shown). These results suggest
that uranium removal by P. aeruginosa CSU occurs in-
dependently of oxygenative respiration or other appar-
ent metabolic activity. Bacterial cell-wall and intracellu-
lar components of P. aerugznosa contain the active sites
for uranium-binding of deposition, which was verified
by electron microscopic observations. Intracellular ura-
nium deposition in P. aeruginosa living cells has been
reported previously.26
Interference of Cations and Anions
on Uranium Sorption
Preliminary experiments have been conducted to deter-
mine the effect on uranium binding by P. aeruginosa
CSU of metals also present in authentic waste streams.
Iron( 11), iron( 111), copper( 11), aluminum( 111), chromi-
um( 111), and lead( 11) inhibited uranium binding (Table
11). The order of inhibition to uranium binding was Fe3+
> Fez+> A13+> Pb2+> Cu2+> C? > Cd2+Mn2+,Ba2+,
Co2+.Iron(II1) caused a severe abatement of uranium

Table I. Effect of various pretreatments on uranium biosorption by Pseudomonm aeruginosa CSU.

~~~

Heat treatment Inorganic treatment Organic treatment Other treatment

URa URb UR" URd URC
Treament (%I (%I Reagent (%I Reagent (%I Reagent (%I
None 92.7 95.4 Water 47.1 Water 47.1 Water 38.8
Oven heat (llO"C, 4 h) 93.3 94.9 0.1 mN HCI 49.0 Diethyl ether 81.7 SDS (0.1%) 40.6
Autoclave (30 min) 74.3 78.0 50 mN HCI 45.8 Ethanol (95%) 83.4 Triton XlOO (0.1%) 35.8
Boil (30 min) 77.7 89.8 0.1 mN H N 0 3 48.6 Methanol 90.9 CHAPS (0.1%) 34.9
Boil (10 min) 72.7 89.3 50 mN N H 0 3 42.4 Methylene chloride 94.3 Alkaline phosphatase 35.9
0.1 mN H2S04 49.9 Dioxane 95.0 Lipase (1 mg/mL) 43.4
50 mN H2S04 36.4 Acetone 97.9 Lysozyme (1 mg/mL) 39.4
0.1 mN NaOH 28.6
50 mN NaOH 28.8
3% (v/v) H203 58.4

"100-ppm initial uranium concentrations; 30-min contact. UR = uranium removal as a percentage of total uranium in solution.
blOO-ppm initial uranium concentrations; 4-day contact.
Treatment for 30 min at 22°C; 95-ppm initial uranium concentrations; 2-h contact.
dTreatment for 30 to 60 min at 22°C; 95-ppm initial uranium concentration; 2-h contact.
eTreatment for 30 to 60 min at 22°C; 100-ppm initial uranium concentration; 2-h contact.

HU ET AL.: BIOSORPTION OF URANIUM BY P. AERUGlNOSA 241

Table II. Effect of coexisting cations on uranium biosorption by 70 I 1 I
Pseudomonas aeruginosa CSU.
Relative uranium Relative uranium
1
Cation removal (%)" Cation removal (%)

Control (UO:+) (100.0) COZt 95.6

H+ 107.5 Ba2+ 95.2
CS' 99.9 Mn2+ 95.1
Li+ 99.7 Cd2+ 93.4
Na+ 98.6 cu2+ 74.0
Kf 98.6 Pb2+ 65.1
NtLt 96.8 Fez+ 41.3
Mg2+ 100.0 Fe3 15.1
Ca2+ 99.0 C13' 75.6
NiZ+ 97.4 AI3 46.9 1
aDefined as (% U removal in presence of cation/% U removal
in control).

removal, which is consistent with the competition be-

tween the two metals for reducing equivalents (Figs. 3
and 4). P. aeruginosa CSU showed a higher specificity
for iron(II1) than for uranium(V1) (Fig. 3). When the
Fe( III)/U( VI) ratio was greater than approximately 6,
the U(V1) binding was completely inhibited, and all the
active binding sites on the biomass were saturated or
shielded by the Fe(II1). Conversely, at low Fe(III)/ Parallel Testing of P. aeruginosa CSU Biomass
U( VI) ratios, the presence of uranium inhibited the with Other Sorbents
Fe(II1) binding (Fig. 4). Thus, the nature of U(V1) and
Comparisons of P. aeurginosa CSU biomass with ion-
Fe(II1) binding to the biomass was competitive.
exchangekhelating resins for uranium removal from
Experiments were also designed to study the possible
pure uranyl nitrate solution are shown in Figure 5. AG
interfering effect of the sulfate group (SO;-),which is
50W-X4, Dowex SOW-X12, and Chelex 100 (Na form)
present in authentic wastewater, on uranium sorption
resins showed superior uranium loading performance.
by the CSU biomass. The results showed no significant
However, P. aeruginosa CSU biomass showed loading
inhibition effect due to the presence of sulfate even at
results [2.13 mg U/g (dry weight)] comparable to those
concentrations as high as 30,000 mg/L (Table 111).
for AG 50W-X4 resin [2.19 mg U/g (dry weight)] when
tested with low-level uranium (11.7-ppm) solutions. Al-
though inorganic resins had a maximum uranium load-
;11
c
1 ing close to that of P. aeruginosa CSU biomass, their
affinity for uranium (at low level) was much lower than
the latter. In addition, an anion exchange resin, Dowex
U(V1) preference area 21K (16 to 20 mesh), was chosen for testing of uranium
/
/ removal under low-pH (2.5) conditions. Almost none
.- I 1
/
s
of the uranium was removed from the solution when
f 7 c / the initial uranium concentration was 0 to 1500 ppm,

Table 111. Effect of sulfate on uranium biosorption by P. aerugi-

nosa CSU."
~~

sL- , 1
3
-
Fe(II1) preference area
i soj- conc.
(PP4
Uranium loading capacity (mg U/g dry weight)

With Na2S04 With equivalent Na from NaN03

/

.-B 2- /' 0 71.3 71.3

.-a 1000 74.2 78.3
/ 80 5000 70.6 73.7
W " 0 U ' ' 1 I 1 ' ' ' ' ' '
0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 10,000 69.6 84.3
Initial U(vr)/Fe(lll) concentration ratio in solution 30,000 68.5 81.5

Figure 3. Selectivity of Pseudomonas aeruginosa CSU biomass be- aInitialuranium concentration 100 ppm; pH 2.5; 1mg CSU biomass/
tween U(V1) and Fe(II1) at pH 2.5 and 22°C. mL: 22°C.

242 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 51, NO. 2, JULY 20, 1996
 25
350 I I
20

:I[
00

2.5,
I&
0 1
,
2
,
3
,
4
,
5
,
15

10

05

00
0
2.5,
1
,
2
,
3
,
4
I
5
I ,
2.0

1.5

10

Uranium in liquid phase (mg/L) 0.5

Figure 5. Parallel testing of P. aeruginosa CSU biomass with ion- 0.0

exchangekhelating resins for uranium removal from a pure uranyl
nitrate solution at pH 2.4. (0) Activated carbon pellets (type “KE,”
6*14 mesh); (V)Chelex 100 resin (Fe form, 100 to 200 mesh, from
Bio-Rad); (0) Amberlite IRC-718 resin (from Supelco); (H) Duolite
C-467 resin (Na form, from Supelco); (V) Chelex 100 resin (Na form,
100 to 200 mesh, from Bio-Rad); ( 0 )Dowex 5OW-X12 resin (H form,
200-400 mesh, from Bio-Rad); (A) AG 50W-X4 resin (H form, minus
400 mesh, from Bio-Rad); (+) P. aeruginosa CSU biomass (lyophi-
lized); ( 0 )Inorganic resin (titaniummonohydrogenphosphate-based,
500 to 600 mesh, from Dr. J. L. Collins); (A) Inorganic resin [potas-
sium, cobalt(11), and hexacyanoferrate(11) composite entrapped in
titanium phosphate matrix, 500 to 600 mesh, from Dr. .I. L. Collins].
indicating that uranium species at such low pH levels
exist primarily in cationic complex forms.
Data from subsequent parallel tests of P. aeruginosa
CSU with one ion-exchange resin (AG 50W-X4) and
one chelating resin (Chelex 100, Na form) in the pres-
ence of Fe3+,AP+,Ca2+,and Mg2+are graphed in Figure
6. Although AG 50W-X4 showed significant uranium
removal from pure uranyl nitrate solution, each of the
four cations inhibited its uranium-binding ability. P.
aeruginosa CSU showed equivalent resistance to the
uranium-binding inhibition by A13+, Ca2+,and Mg2+;
however, Fe3+ inhibited all the uranium-binding sor-
bents tested here. Preliminary experiments were also
carried out for uranium removal from 5% sodium car-
bonate solution (100 ppm U), in which uranium exists
in anionic uranium-carbonate complex forms. The ura-
nium loadings were 26.6,23.0,and 24.0 mg/g dry weight
for P. aeruginosa CSU, AG 50W-X4 resin, and Chelex
100 resin (Na form), respectively.
Results of parallel tests of P. aeruginosa CSU biomass
and the biosorbents obtained from Dr. Volesky for ura-
nium removal both in and without the presence of Fe3+
are shown in Table IV. P. aeruginosa CSU demonstrated
the best capability of uranium removal and resistance
to ferric iron inhibition, particularly under low levels
of initial uranium concentrations. Dissolution of alga-
based biosorbents (VS#2 and VS#5, without cross-
linkage) in the acidic solution has been noted, although
0 1 2 3 4 5
Metal-ion concentration (g/L)
Figure 6. Comparison of uranium removal from dilute (10-ppm)
P.aeruginosa CSU
solutions in the presence of other metal ions. (0)
biomass; (A) Chelex 100 resin (Na form); (0) AG 50W-X4 resin
(hydrogen form).
each showed good uranium sorption capability. Com-
parisons of VS#2 with VS#3 and VS#5 with VS#6 indi-
cated that chemical cross-linking processes cause some
loss of uranium-binding sites.
Effect of Operating Conditions on Uranium
Sorption by P. aeruginosa CSU
As shown in Figure 7, the CSU biomass removed ura-
nium from solution very rapidly and 99% of the sorption
equilibrium should be reached in minutes. The pH of the
solution significantly affected both the uranium sorption
dynamics and the equilibrium uranium loading capacity
(Fig. 8), while temperature did not. When the pH was
below 5, uranium removal was due to the biomass bind-
ing rather than chemical precipitation; with increasing
pH, uranium removal by the CSU biomass increased
more than twofold (Fig. 8).
DISCUSSION
Pseudomonas aeruginosa is a common bacterium found
in soil and water. It is a Gram-negative, aerobic rod (0.5
to 0.8 pm by 1.5 to 3.0 pm) that is known for its ability
to utilize many carbon sources. P. aeruginosa CSU, ob-
tained from the ORNL culture collection, was originally
isolated by H. R. Meyer and S. Johnson at Colorado
State University from an aquatic system of the Rocky
Flats en~ironment.’~ Use of this bacterium for uranium
bisorption was first reported by Shumate et alF7; also,
additional, but inadequate, data were reported in a later
study.26The work described in the present article was

HU ET AL.: BlOSORPTlON OF URANIUM BY P. AERUGlNOSA 243

 Table IV. Parallel testing of P. aeruginosa CSU with other biosorbents for uranium removal
from acidic solutions and competition by ferric iron.

Uranium removal (%) Inhibitionb (%)

Oppm 10ppm 100ppm Oppm 10ppm 100ppm

Biosorbent" Fe3+ Fe3+ Fe3+ Fe3+ Fe3+ Fe3+
P. aeruginosa CSU 82.4 49.7 17.8 0.0 39.7 78.4
VS#1 20.9 1.9 1.9 0.0 90.9 90.9
VS#2 55.5 5.6 5.1 0.0 89.9 90.8
VS#3 36.0 4.0 5.9 0.0 88.9 83.6
VS#4 25.2 3.4 1.4 0.0 86.5 94.6
VS#5 67.9 17.5 10.9 0.0 74.2 84.0
VS#6 65.3 24.7 19.2 0.0 62.2 70.6
VS#7 29.5 9.1 3.2 0.0 69.2 89.2
VS#8 37.0 15.8 4.0 0.0 57.4 89.2

"VS#l = biosorbent based on an industrial fungus, Ostsorb L9; VS#2 = marine algal
biomass, A. nodosum #5 (0.841 to 1 mm); VS#3 = biosorbent based on a marine alga, A.
nodosum #5, formaldehyde in HCl, cross-linked; VS#4 = biosorbent based on an industrial
fungus, R. nigricans, cross-linked in polyethyleneimine (PEI) and glyoxal mixture; VS#5 =
marine algal biomass, Sargassum #5; VS#6 = biosorbent based on a marine alga, Sargassum
#5, cross-linked in PEI and glutaric dialdehyde; VS#7 = biosorbent based on an industrial
fungus, Rhizopus nigricans #3 (China, 0.295 to 0.5 mm, washed); VS#8 = industrial fungus
biomass, P. chrysogenum #3 (China, 0.295 to 0.5 mm, washed).
bInhibition percentage was calculated relative to the uranium removal percentage in the
absence of ferric iron.

performed to provide further characterization of P. aer-
uginosa CSU relative to its uranium-binding properties.
Instead of YM media, use of defined growth medium
(M9) in conjunction with an iron-deficient condition
improved the uranium loading from approximately 100
to 200 mg U/g dry CSU (data not shown). Successful
scale-up of P. aeruginosa CSU cultivation from a
4-L flask to a 500-Lstirred-tank fermentor validated
its feasible production on an industrial scale. Cells of
different ages collected before, during, and after mid-
log growth phase, as well as fresh or old resting cells
stored in the refrigerator, did not show significant differ-
ences in uranium-binding capability, indicating that the
timing in harvesting cells is not critical. Lyophilized P.
aeruginosa cells can retain their uranium-binding prop-
erty after long-term storage at room temperature
(22°C).

-
,
100 100

f- 80 5
- 80 ~

c
.- .-
-5:
3 3
5:.
60- -

.I 60 z
&
d -
-8 70 40
-

a
z
;
E
.-B
40 .Ee 20 - -
3
e
3
0- -
20
I I I I I
0 2 4 6 0 10 12

244 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 51, NO. 2, JULY 20, 1996
 Well-controlled pretreatment of microbial biomass
could play an important role in improving biomass prop-
erties for uranium uptake. The results from both this
work and previous work9were in agreement in that cell
viability has little effect on uranium binding, although
different types of biomass were used for the tests. Dead
biomass offers several advantages in that it is not subject
to metal toxicity or adverse operating conditions, needs
no nutrient supply, and may be regenerated by relatively
simple nondestructive treatment^?^ Thus, inactivated,
nonliving microbial biomass can serve as a basis for
the development of potent biosorbent materials for met-
a l ~The . ~effects
~ of heat and chemical pretreatment for
mycelial Penicillium biomass were reported earlier by
Galun et a1?.z8 The uptake and binding of Ni, Zn, and
Cd by this biomass were enhanced by preheating at
100°C for 5 min and exposure to alkali and dimethyl
sulfoxide (DMSO). Although the maximum quantity
bound remained unchanged, the uptake of Pb by myce-
lial preparations after heat treatment was more rapid."
For U( VI) accumulation by the Penicillium biomass, it
was found that boiling, alcohol treatment, pasteuriza-
tion, and trichloroacetic acid pretreatment do not re-
duce and may actually increase fungal In
the current work, heat pretreatments did not harm and,
in some cases (e.g., autoclaving for 25 min, then baking
at 85°C for 4 h), even enhanced uranium loading perfor-
mance (data not shown). The CSU treatment by organic
solvent seemed to be the most efficient method for en-
hancing uranium loading capacity. This finding sug-
gested a solubilization of cell membranes (outer, cyto-
plasmic, or both) by the solvent; however, treatment of
cells with specific cell envelope reagents, such as lipase,
alkaline phosphatase, lysozyme, etc., had no effect on
uranium removal or binding. The potential for syner-
gism between individual beneficial pretreatments has
not yet been determined.
The model of the multiplicity of uptake sites proposed
by Tobin et al.29330may provide one of the explanations
for the cation competition with uranium biosorption by
P. aeruginosa CSU. Ferric iron inhibition to uranium
bisorption seems to be universal for several biomasses.
For uranium removal from uranyl chloride solution by
the Penicillium biomass,1° Fe3+showed the most inhibi-
tory effect among metal ions such as CrO;-, MOO;-
Niz+,Cuz+,Zn2+,Cdz+,and Pbz+.The remarkable Fe3+
inhibition to uranium biosorption from a process solu-
tion also occurred with Rhizopus arrhizus and Strepto-
rnyces l e v o r i ~Although
.~ the biomass used in our work
(P. aeruginosa CSU) is different from the Penicillium,
Rhizopus arrhizus, or Streptomyces levoris, they all
showed Fe3+preference over uranium relative to bio-
sorption. Additionally, similarities of the effect of ferric
and uranyl ions on P. aeruginosa biological behavior
(i.e., its capability of growth and production of metal-
sequestering agents siderophores) were also rep~rted.'~
These results indicate that Fe3+is an uranium analogue
HU ET AL.: BIOSORPTION OF URANIUM BY f . AERUGlNOSA
that could be biosorbed by uranium-binding sites.
Therefore, in designing a process for wastewater treat-
ment, the step for preremoval of iron seemed to be
essential for uranium biosorption by P. aeruginosa CSU.
For uranium bisorption by R. arrhizus, the pH of waste-
water was adjusted to 4. Removal of the iron due to
precipitation at higher pH levels was beneficial to avoid
strong ferric ion competition with uranium for biosorp-
tion by the b i ~ m a s s . ~
Further, P. aeruginosa CSU has the capability for re-
ducing both ferric iron [Fe3++.Fez++ Fe) and uranium
(U6+(soluble) +.U4+,intracellularly precipitated]. Intra-
cellular deposition of uranium in CSU cells was reported
earlierz6and was also experimentally observed through
transmission electron microscopic studies, although the
actual mechanism for such intracellular deposition
(metal reduction) has not yet been elucidated. When Fe3+
and U6+are present in the same solution, the fact that
iron has a higher redox potential (Fe3++ e Fe*+,Eo =
0.771 V) than that of uranyl ion (UO$+ + 2e U4+,
Eo = 0.327 V)19 may be another interpretation for iron
inhibition to the uranium binding (or uranium intracellu-
lar precipitation), because Fe3+always has the preference
over U( VI) to receive electrons donated from the CSU
biomass. Ferric iron may oxidize the precipitated form
[U( IV)] back into a soluble form [U(VI)]. This is actually
the basis for biological leaching of the ore. It involves
the bacterial oxidation of pyrite to produce an oxidizing
sulfuric acid solution rich in ferric iron, which oxidizes
and solubilizes the uranium in the
The pH profiles (i.e., uranium loading by biomass
increased with increasing pH under acidic to near-
neutral pH conditions) may be explained by the H+
competition with the uranium binding sites, as suggested
earlier by Galun et a1.: who noted that an increased
H+ concentration generally resulted in a suppressed
metal-ion uptake by Penicillium biomass. For Rhizopus
arrhizus and Streptomyces levoris, the metal biosorption
capacities declined substantially under lower pH (or
higher H+) condition^.^.^^ These pH profiles agree
with our results; however, the uranium loading at low
pH levels (e.g., pH 2, approximately 50 mg U/g, dry
weight) for the CSU biomass is better than the one
(30 mg U/g, dry weight) for Rhizopus arrhizus. At lower
pH levels (e.g., pH 2 or 3), the cell walls may be proton-
ated, resulting in a weak complexation affinity between
the cell wall and uranyl cations. Furthermore, the uranyl
ions are highly soluble in an acidic solution, thereby
decreasing biosorption effi~iency.'~ The reduction in
uranium loading capacities with decreasing pH may be
also due to cell structure damage at very acidic condi-
tions; however, definitive results would require further
microscopic studies.
The hydrolysis of uranyl ion may play a significant
role in determining the equilibrium between uranium
in solution and in CSU biomass, The speciation of ura-
nium in solution may also aid in the explanation of pH
245
profiles as shown in Figure 8. When the pH increases
from an acidic value to a neutral value, various uranium
hydroxo complexes may form, the repartition of which
is conditioned by the pH and the total uranium concen-
trati0n.27~~Repartition of these hydroxo complexes is
determined by the following eq~ilibria'~:
UO$+ + 2H20 U02(0H)++ H 3 0 +
(pK = 5.8) (2)
2UO$+ + 4H20 * (U02)2(0H)$+ + 2H30+
(pK = 5.62) (3)
*
3UO$+ + 10H20 (U02)3(OH):+ + 5H30+
(pK = 15.63) (4)
At pH values below 2.5, the predominant species
is UO$+;but at pH values above 2.5, hydrolysis prod-
ucts include U02(0H)+, (U02)2(0H)$+, and
(U02)3(0H)g.34 Above pH 4, the major forms of ura-
nium in solution are cationic hydroxo-complexes. Obvi-
ously, P. aeruginosa CSU biomass can bind these hy-
droxo complexes very well. By comparison with the
control experiments, it is also clear that the higher ura-
nium loading at higher pH levels (but below 5.0) was
not due to the decrease in uranyl solubility at higher
pH levels (Fig. 8). Ionic size may induce biosorption
of hydrolyzed species; thus, the CSU biomass would
remove a greater quantity of uranium.
Unlike nitrate, which is a weak complexing ligand
for U(VI), the sulfate present in the uranium solution
complexes with U(V1) and is in dynamic equilibrium
with uranyl ion^.^,'^,'^
UO$+ + SO$- U02S04 (K1 = 7.9 X lo2) (5)
UO$+ +2SO$- * U02(S04)$- (K2 = 1.5 X lo4) (6)
Unlike the strong inhibition of sulfate to the uranium
biosorption by fungal biomass$30 our results did not
show significant sulfate inhibition to uranium biosorp-
tion by CSU biomass. This may indicate that CSU binds
UO$+and is strong enough to drive the above equilib-
rium to the left side. It is also possible that CSU could
contain sites that bind uranium in the form of anionic
sulphato complexes. This may be supported by the data
shown in Figure 8, indicating that, at the lower pH end
of the pH profile, the presence of sulfate helped remove
more uranium from solution by the biomass as com-
pared with the parallel test with only nitrate as the
solution background.
Finally, to optimize uranium biosorption by CSU bio-
mass, further information is needed concerning the solu-
tion chemistry of uranium at different pH levels and
the effect of the presence of other ions or components.
In addition, studies of cell-wall chemistry (functional
groups contributing to uranium binding) and of the
mechanism of intracellular uranium deposition are re-
quired. Uranium solution chemistry plays an important
part in the biosorption mechanisms; for example, at
different pH levels, the repartition of uranyl species
246 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 51, NO. 2, JULY 20, 1996
changes and influences the nature, charge, and size of
ions able to be complexed, adsorbed, and accumulated
by the biomass.14
CONCLUSIONS
In the tests using pure uranyl nitrate solution at low-
pH conditions (pH 2.5), P. aeruginosa CSU showed the
highest uranium biosorption capability (approximately
100 mg U/g dry biomass) among the candidates tested
in this study. From low to near-neutral pH conditions,
the uranium loading capacity of P. aeruginosa CSU in-
creased with increasing pH, up to the point where pre-
cipitation began to occur. Both cell-wall chemistry and
uranium solution chemistry play important roles in con-
trolling the bisorption phenomenon. The lyophilized
CSU biomass was very stable when stored at room tem-
perature. Little loss of uranium binding activity was
noted when the biomass was treated under harsh condi-
tions (high temperature, in acid and base solutions, or
in surfactants and enzymes). Twofold enhancement of
uranium binding was achieved by pretreatment of cells
with polar organic solvents such as acetone. A study of
the competition of cations with uranium for biosorption
by CSU showed that ferric iron significantly inhibited
uranium binding. Ferric iron is such a uranium analogue
that will inhibit uranium binding to various biomasses.
Unlike other biomasses reported in the literature, the
presence of sulfate did not appear to pose a problem
for uranium removal from wastewater by CSU. The
parallel testing of CSU biomass with other biosorbents
and ion-exchangekhelating resins in and without the
presence of ferric iron interference suggests that CSU
is a competitive and potential candidate for the restora-
tion of acidic wastewaters, based on both technical and
economical considerations. However, for efficient use
of CSU biomass, a preprocessing step that removes most
of the ferric iron from the wastewater seems to be nec-
essary.
The authors thank Dr. J. L. Collins, R. E. Ihli, and S. S.
Laughlin for their help with the experimental work, and Dr.
B. H. Davison for his helpful comments and technical review
of this manuscript. The financial support of the US. Depart-
ment of Energy, Office of Technology Development, is also
gratefully acknowledged. Michael 2.-C. Hu is a postdoctoral
fellow appointed to the Oak Ridge National Laboratory
Postdoctoral Research Associates Program administered
jointly by the Oak Ridge Institute for Science and Education
and the Oak Ridge National Laboratory.
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OF URANIUM BY P. AERUGlNOSA 247