Appl Microbiol Biotechnol (1989) 31:613-618
Microbiology
Adsorption of mixed metals and cadmium
by calcium-alginate immobilized Zoogloea ramigera
Sabine P. Kuhn and Robert M. Pfister
Department of Microbiology, The Ohio State University, Columbus, Ohio 43210, USA
Summary. Zoogloea ramigera 115 cells were im-
mobilized into beads of calcium alginate and used
in air-bubbled column reactors to remove cad-
mium (Cd), zinc (Zn), manganese (Mn), lead (Pb),
copper (Cu) and strontium (Sr) from dilute and
concentrated solutions. By placing three bubble
columns in sequence it was possible to achieve Cd
adsorption efficiencies of 99% or greater. During
ten applications of approximately 100 lxg-m1-1
Cd to three reactors in sequence, the immobilized
Z. ramigera adsorbed 99.9% of the metal. The ef-
ficiency of the first column decreased from 92.2%
on the first day to 53.8% on the tenth day, but the
overall efficiency remained high because of the
other two reactors. Exposure of bubble columns
to mixed metal solutions yielded similar results.
Cd, Zn, Mn, Cu, and Sr were adsorbed at 95.9%
or better when concentrations in the mixtures
ranged from 12 to 117 Ixg-ml71 per metal. Pb was
the least efficiently adsorbed metal with 81.3%
and 74.7% removed from solutions containing 12
and 23 ~tg-m1-1, Pb respectively. Recovery and
concentration of the metals was possible with ni-
trilotriacetic acid (NTA) treatment; which de-
sorbed > 77% of the accumulated Cd from the im-
mobilized cells. Recovery of metals from beads
which had been exposed to mixed metals de-
pended on the metal; only 5.8% of the adsorbed
Sr was recovered, but 98.3% of Zn was desorbed.
Introduction
Zoogloea ramigera 115 is a Gram-negative bacte-
rium known for its extracellular weakly acidic
Offprint requests to: R. M. Pfister
@pried
Biotechnology
© Springer-Verlag 1989
carbohydrate polymer (Friedman and Dugan
1968; Tezuka 1973; Unz and Farrah 1976). This
polysaccharide polymer acts as a polyelectrolyte
and adsorbs such metal ions as Co 2+, Cu 2+,
Cd 2+, UO22+, and Fe 3+ (Norberg and Persson
1984; Parsons and Dugan 1971). Adsorption of
metal cations is possible through phosphoryl, car-
boxyl, sulphhydryl and hydroxyl groups which
can be found on cell wall components, proteins
and lipids (Jellinek and Sangal 1972; Rendelman
1978; Steiner et al. 1979). Metal adsorption by Z.
ramigera is attributed mainly to its extracellular
polymer. The polymer, which is composed of D-
glucose, D-galactose and pyruvic acid in an ap-
proximate molar ratio of 11:3:1.5 should allow
abundant binding due to its slight negative charge
and its many hydroxyl groups (Ikeda et al.
1982).
Attempts have been made to use the metal-
binding capacity of microorganisms such as bac-
teria, yeasts and algae, for the clean-up of indus-
trial effluents. Norberg and Persson (1984) floccu-
lated dead Z. ramigera to remove Cd 2+, Cu 2+
and UO 2+ from solutions. Citrobacter sp. has
been studied most extensively in Cd removal. The
bacterium is immobilized in polyacrylamide and
removes Cd when glycerol 2-phosphate is in-
cluded in the metal-containing solutions. The sub-
strate induces a cell-bound phosphatase which
precipitates the metal on the bacterial cell surface
as metal phosphate (Macaskie and Dean 1984a,
b). We set out to develop a way in which to use Z.
ramigera to clean up industrial effluents. For a
biological metal removal system to work on an in-
dustrial scale, the organism used has to be readily
contained and the set-up has to be reusable. Here
we describe adsorption capacity experiments to
characterize metal adsorption by immobilized Z.
ramigera.
614 S.P. Kuhn and R. M. Pfister: Adsorption of heavy metals by immobilized Z. ramioera
Materials and methods
Strain and media. Zoogloea ramigera 115 (ATCC 25935) was
obtained from The Ohio State University Culture Collection,
Columbus, Ohio and grown in either CY medium [5 g/l casi-
tone (Difco, Detroit, Ill), 1.0 g/1 yeast extract (Difco), modif-
ied from Unz and Farrah 1972] or a defined medium (DM,
Parsons and Dugan 1971) containing 0.5 g/1 arginine-HC1
(Sigma Chemical Co., St. Louis, Mo, USA), 1,0 g/l alanine
(Sigma), 0.2 g/1 MgSOa.7H20, 2.0 g/1 K~HPO4, 1.0 g/1
KHzPO4, 20 g/1 glucose [Cerelose-Dextrose 2001 (CPC Inter-
national, Englewood Cliffs, N J, USA)] and 0.002% yeast ex-
tract (instead of vitamin B12 used by Parsons and Dugan 1971).
The organism was grown on a rotary shaker at 28 ° C. Media
were inoculated with 0.1 ml from stock cultures (approxi-
mately 109 cells/ml) stored in liquid nitrogen.
Atomic absorption spectrophotometry (AAS). Metals were quan-
tified using a Perkin-Elmer atomic absorption spectrophoto-
meter Model 403 (Norwalk, Conn, USA) equipped with an air-
acetylene flame and the appropriate single-element lamps.
Immobilization of Z. ramigera. Zoogloea ramigera was cul-
tured overnight in 500 ml CY or DM medium at 28 ° C, har-
vested by centrifugation at 10400 g for 10 min and the pellet
was suspended in 20 ml of 0.85% NaC1. The resuspended cells
were added to 250 ml of 2% high viscosity sodium alginate
(Sigma, final concentration: 1.85%) in 0.85% NaC1 and mixed.
The cells were immobilized by forcing the cell-alginate mix-
ture through 25 gauge needles into 250 ml of 1.47% CaC12 so-
lution using a variable speed pump. The beads so formed,
were allowed to harden for 2 h, stabilized with 250 ml of 1%
polyethyleneimine (PEI), pH 5.0, (Polysciences, Warrington,
Pa, USA), in double distilled H20 (ddH20), for 5 min and
washed with 3 1 ddHzO. Beads of alginate alone were formed
as those containing cells, except that 20 ml of 0.85% NaC1
without cells was added to 2% alginate. After stabilization the
immobilized cells were placed into a bubble column reactor in
CY or DM medium for 18-24 h, before exposure to solutions
containing metals. Solutions were not buffered and were made
from concentrated stock solutions of the metal chlorides, ex-
cept for ZnSO4.7H20 and Pb(NO3)2.
All experiments with immobilized cells were carried out at
room temperature. Zoogloea ramigera used for immobilization
was grown at 28 ° C, but growth of imobilized cells was at room
temperature.
Bubble column reactor. A glass tube (diameter, 9 cm; height, 29
cm) containing a ground glass filter at the bottom and sealed
with a rubber stopper at the top was used for all experiments.
A hollow rod, covered with nylon netting, was inserted
through the stopper to reach the bottom of the column. This
tube was used to pump liquid in and out of the reactor. The
column was aerated at 250-350 cc/min, with compressed air
forced through the ground glass filter at the bottom of the
reactor. All volumes for metal exposure, washing and recovery
with nitrilotriacetic acid (NTA) were arbitrarily set at 500 ml.
Although the capacity of each column was approximately 2 1,
the working volume was generally 750 ml (500 ml of liquid
and 250 ml of beads). Larger volumes were not used due to
problems with foaming and pressure build-up.
Sequential reactors. Three times 500 ml of Z. ramigera were
grown in DM medium for 18-24 h, the harvested cell pellets
were combined, resuspended in 60 ml 0.85% NaC1 and 20 ml
was added to three times 250 ml of 2% alginate. The cell-algi-
nate mixtures were then used in immobilization to generate
three batches of beads. Following immobilization procedures
each set of 250 ml beads was placed into a bubble column
reactor in 500 ml DM medium for 24 h. At that time the me-
dium was pumped out and the beads were washed with 500 ml
ddH20. The first reactor (R1) was exposed to a metal-contain-
ing solution for 30 min and a 3 ml sample was taken every
10 rain. That solution was then pumped out of the reactor and
replaced with 500 ml ddH20. The metal solution removed
from R1 was added to the washed beads in the second reactor
(R2) for 30 min, and sampled every 10 min. After 30 min the
solution was pumped from R2, replaced with ddH20, and ad-
ded to the third reactor (R3). Samples were taken for 30 min
and the solution left in contact with beads in R3 until the ex-
posure process was repeated the next day.
Exposure to 100 #g. ml- 1 Cd. Immobilized cells in columns 1-
3 were exposed to Cd-containing solutions after initial over-
night growth every day for 10 days as described above. After
10 days the immobilized cells in the first column no longer
adsorbed as much metal. To prevent a decrease in overall me-
tal adsorption efficiency, the beads in the first reactor were
treated with 0.48% NTA, pH 6.0, to recover adsorbed Cd. Fol-
lowing NTA exposure the immobilized cells were placed into
Z. ramigera-seeded DM medium (inoculum was grown at
28°C for 18 h). After 18 h, the regeneration was complete, the
medium was removed, the beads washed, and the Cd exposure
regime was continued for another 10 days.
Exposure to mixed metals. The basis of this experiment fol-
lowed the premise of the Cd sequential reactors. However, the
beads in the first column were exposed to four metal solutiGns
containing a total of 72, 138, 360, and 702 Ixg. m l - 1 Cd, Pb, Sr,
Cu, Zn, and Mn (12, 23, 60, and 117 Ixg.ml -~ of each individ-
ual metal). The beads in columns 2 and 3 received solutions
which had previously been in R1 or R2 for 30 min, respective-
ly. Exposure time was 30 min per reactor. Three milliliter sam-
ples were removed at 10 rain intervals. The metal solutions
pumped were replaced with ddH20.
Recovery of metals with NTA. Cd was recovered from the im-
mobilized cells in the first reactor after 10 days of metal expo-
sure using 500 ml of 0.48% NTA, pH 6.0, for 3 h. The first co-
lumn, containing beads exposed to four multiple metal solu-
tions, was treated the same way. To better understand the ac-
tion of NTA, immobilized cells were pretreated with 0.24%
NTA, pH 6.0, or exposed to a solution containing both 50
Ixg. ml-1 Cd and 0.24% NTA, pH 6.0.
Results
The literature indicates that Z. ramioera will re-
move Cd and other metals from solutions (Nor-
berg and Persson 1984; Parson and Dugan 1971).
Our experiments were designed to make use of
these metal-binding properties by immobilizing Z.
ramigera in Ca-alginate beads. Immobilization
did away with the need for centrifugation or floc-
culation (Norberg and Persson 1984) since the
beads settled quickly. Results from our laboratory
(to be published elsewhere) had established that
the pH optimum for metal exposure was between