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Appl Environ Microbiol, April 1998, p. 1545-1547, Vol. 64, No. 4
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Effects of Cellular Metabolism and Viability on
Metal Ion Accumulation by Cultured Biomass from a Bloom of the
Cyanobacterium Microcystis aeruginosa
D. L.
Parker,1,*
L. C.
Rai,2
N.
Mallick,2
P. K.
Rai,2 and
H. D.
Kumar2
Department of Biology and Microbiology,
University of Wisconsin Oshkosh, Oshkosh, Wisconsin
54901,1 and
Department of Botany,
Banaras Hindu University, Varanasi 221005, India2
Received 24 July 1997/Accepted 15 January 1998
 |
ABSTRACT |
The sorption of nickel, cadmium, and copper by cultured biomass
from a naturally occurring bloom of Microcystis aeruginosa was demonstrated in two systems: cells suspended in culture medium and
cells immobilized in alginate. Incubation in the absence of light, in
the presence of metabolic inhibitors, and at 4°C did not
substantially decrease the copper accumulation by cells in culture
medium. Heat-killed, formaldehyde-treated, and air-dried biomass
samples sorbed nearly as much (or in some cases slightly more) copper
as did viable samples.
| |
TEXT |
The recovery of valuable or toxic
metals by biological methods, attractive alternatives to conventional
physiochemical processes, is finding significant application in various
remediation strategies (3, 6, 7, 13, 26, 27, 29). Metal
sequestration by organisms is often termed biosorption, which includes
both passive adsorption of metals to cell envelopes and metabolically mediated uptake (4, 8, 12, 22, 23, 29). The nature of the
biosorption depends on the metal and the microorganism (3, 5, 9,
23).
The cyanobacteria Microcystis aeruginosa and
Microcystis flos-aquae exist in multicellular aggregates
which are surrounded by a colonial capsule or exopolymeric matrix that
can adsorb substantial amounts of various metal ions (1, 17, 20,
21). Metal accumulation by Microcystis has been
reported for field samples (1, 10, 11, 25), axenic cultures
(17, 18, 20, 21), and purified exopolymers (2, 17, 20,
21).
Growths of M. aeruginosa in Laxmi Pond (25°20'N,
83°0'E), Varanasi, India, have been studied at frequent intervals for
several years (19). M. aeruginosa was
consistently the dominant phototroph in this pond (19),
which typifies a prevalent kind of subtropical body of water. The
sequestration of metals by Microcystis in these ponds has
numerous ecological and public health implications. Furthermore,
similar ponds (including ones created in alkaline and saline
wastelands) are potentially suitable for the mass cultivation of
cyanobacteria and the harvesting of their products.
A procedure for obtaining large amounts of biomass from cyanobacterial
blooms was developed with the goal of investigating the metal binding
potential of material from natural growths. Surface samples from dense
blooms in which M. aeruginosa was the only detectable
phototroph were collected at 2 m from the shore of Laxmi Pond
(19). The pH range of the sampled water was 8.9 to 9.2. The
Microcystis colonies were concentrated by filtration through
100% polyester fabric (Gloria weave; Bombay Dyeing Corp.), suspended
in a 15-fold excess (vol/vol) of deionized water, held for 2 h at
ambient temperature, and collected by filtration. This washing
procedure was repeated twice. The washed cells were cultured under
standard conditions, which were 4 days of growth in DP medium (19) at 29 ± 2°C with 2,100 lx of continuous
irradiance. The pHs of the cultures at 4 days of growth ranged between
8.8 and 9.1. At the beginning of each metal uptake experiment, the
4-day-old culture was diluted appropriately in DP medium, adjusted to
pH 9 or 9.1, assayed (14) for chlorophyll 
(CHL), and
supplemented with a designated metal chloride. Deionized (18 M
)
water and acid-soaked, low-metal plastics (or occasionally acid-leached glassware) were used for all experiments.
Metal sorption by biomass in a liquid medium.
Four-day
cultures in DP medium alone (no metal added) or in DP medium containing
7.87 or 31.5 µM CuCl2 (0.5 or 2.0 µg of Cu per ml),
4.45 µM CdCl2 (0.5 µg of Cd per ml), or 8.52 µM
NiCl2 (0.5 µg of Ni per ml) were incubated under the
standard growth conditions described above. At timed intervals after
the addition of the metal, duplicate 10-ml portions of each culture
were collected on nylon filters (5-µm pore size; Micron Separations,
Inc. Westboro, Mass.) and washed twice with 5 ml (each) of deionized
water. The filtered cells were digested by the method described by
Martin (16). The digested cells and corresponding culture
medium samples were resuspended in 1% HNO3 and analyzed
for metals in a Perkin-Elmer 2380 atomic absorption spectrophotometer.
Steady-state biosorption of cadmium, nickel, and copper was achieved at
2 h of incubation (Fig. 1). Figure
2 summarizes the concentrations of sorbed
and free copper that were observed at 2 h of incubation in
reaction mixtures containing various concentrations of copper (7.87 to
31.5 µM CuCl2) and M. aeruginosa biomass (0.68 to 1.8 mg of CHL per liter). The arrow in Fig. 2 indicates the concentrations of sorbed and free copper at 2 h in subsequent experiments in which the effects of metabolic inhibition on metal accumulation by M. aeruginosa were examined.
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FIG. 1.
Time course of metal sorption by cultured cells from an
M. aeruginosa bloom. (A)  , 31.5 µM CuCl2
(2.0 µg of Cu2+/ml);  , 7.87 µM CuCl2
(0.5 µg of Cu2+/ml); cells at a density of 1.5 mg of CHL
per liter. (B)  , 4.45 µM CdCl2 (0.5 µg of
Cd2+/ml);  , 8.52 µM NiCl2 (0.5 µg of
Ni2+/ml); cells at a density of 1.8 mg of CHL per liter.
Error bars indicate the ranges of duplicate measurements.
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FIG. 2.
Relationship between bound and free copper at various
concentrations of added copper (7.87 to 31.5 µM) and various biomass
concentrations (0.68 to 1.8 mg of CHL/liter), for the steady-state
conditions observed after 2 h of incubation at 29°C in culture
medium. The arrow indicates the conditions of bound and free copper in
subsequent studies of metabolic inhibition.
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|
Effects of metabolic inhibition.
A 4-day culture was adjusted
to 0.86 mg of CHL per liter of DP medium (pH 9.1). Culture aliquots
were pretreated by incubation for 2 h as follows: (i) at 29°C
and 2,100 lx; (ii) at 29°C in the dark; (iii) at 4°C in the dark;
(iv) with 10 µM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) at
29°C and 2,100 lx; (v) with 0.1% formaldehyde at 29°C and 2,100 lx; and (vi) with 2.0% formaldehyde at 29°C and 2,100 lx. Another
aliquot was heated at 80°C for 3 h and then cooled to 29°C.
Each suspension was brought to 7.87 µM CuCl2 and then maintained under the pretreatment conditions, except that the 80°C-heated sample was held at 29°C and 2,100 lx. At 2 h,
duplicate 10-ml aliquots from each culture were filtered, washed, and
assayed for biosorbed metal as described above.
In all cases, the sorption of copper by the variously inhibited or
killed M. aeruginosa cells was at least 84% of that
observed for metabolically active cells (Table
1). This observation is noteworthy
because nonviable material may be safer and more convenient than viable
material in various biotechnological applications.
The largest decrease in copper sorption occurred with relatively gentle
metabolic inhibition, such as incubation with the photosynthesis
inhibitor DCMU (Table 1). In contrast, cells exposed to the most harsh
conditions, i.e., treatment with 2% formaldehyde, actually sequestered
more copper than did viable cells (Table 1), supporting the reports of
other investigators (28) who suggest that formaldehyde
increases metal adsorption by modifying cellular structures or exposing
intracellular binding sites.
Metal sorption by alginate-immobilized biomass.
A portion of a
4-day culture was collected on 5-µm-pore-size filters and dried for
36 h at 45°C with blowing air. A parallel portion of moist cells
was maintained in DP medium under standard growth conditions. The dried
biomass was suspended in DP medium to a concentration of 2 mg/ml (3.5 µg of CHL per ml), as was the moist biomass. Duplicate 2.5-ml
portions of each sample were mixed with 2.5 ml of 10% (wt/vol)
alginate and added dropwise from a syringe into 0.2 M
CaCl2. The alginate beads were washed with DP medium. Each
5-ml aliquot of beads was agitated for 2 h in 50 ml of DP medium
containing 7.87 µM CuCl2. Digestion of the beads and
metal analysis by atomic absorption spectrophotometry have been
described previously (15, 24). Background metal uptake by
the alginate alone was less than 25% of that of any biomass-containing
sample and was subtracted from the data.
For immobilized moist biomass, the initial amounts of metal sorption
during the first 2 h of the reaction were, per mg of CHL per h,
3.56 µmol of copper, 1.88 µmol of cadmium, and 4.94 µmol of
nickel. For immobilized dried biomass, these values were, per mg of CHL
per h, 2.28 µmol of copper, 1.36 µmol of cadmium, and 3.58 µmol
of nickel. The efficacies of Cu, Cd, and Ni sorption by the dried
biomass were, respectively, 65, 72, and 73% of those of moist biomass.
The activity of the dried biomass may have been influenced by both its
clumping and its presumably altered metabolic activity.
In summary, this study suggests that copper accumulation by M. aeruginosa-dominated biomass is largely independent of the metabolic state of the cells. Furthermore, heat-killed, HCHO-treated, and air-dried biomass samples exhibit substantial sorption of copper,
cadmium, and nickel and thus may be promising materials for metal
reclamation technologies. Since naturally occurring M. aeruginosa can exhibit various metabolic states, the surprisingly minor effects on copper sorption that we observed for diverse inhibitory treatments of field-collected M. aeruginosa
biomass are of interest for environmental modeling.
| |
ACKNOWLEDGMENTS |
This research was supported by grants from the Department of
Biotechnology, Ministry of Science and Technology, and the Council of
Scientific and Industrial Research of the government of India to
L. C. Rai and H. D. Kumar and from the Council for
International Exchange of Scholars (Fulbright Program) and the U.S.
National Science Foundation, South Asia Program, to D. L. Parker.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biology and Microbiology, University of Wisconsin, Oshkosh, WI 54901. Phone: (920) 424-3077. Fax: (920) 424-1101. E-mail:
parker{at}uwosh.edu.
| |
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Appl Environ Microbiol, April 1998, p. 1545-1547, Vol. 64, No. 4
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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