Biosorption of cadmium by microorganisms
Duraisamy Prabha*, Subpiramaniyam Sivakumar**, Chandra Venkatasamy
Subbhuraam*
*Department of Environmental Sciences, Bharathiar University, Coimbatore 641 046, Tamil Nadu, India
([email protected])
**Department of Environment and Health, Kosin University, Young Do Gu, Busan 606 701, Republic of Korea
([email protected]).
Abstract: The effects of Cd in aqueous solution on the growth and accumulation properties of bacteria- Bacillus
spp. – B. simplex, B. firmus and B. ehemensis and the fungi, Aspergillus spp. – A. niger, A. glaucus and A. wentii
isolated from Cd amended soil was assessed. Cadmium affected the growth and accumulation properties of the all
selected species of bacteria and fungi in aqueous medium. Bacillus firmus for bacteria and Aspergillus niger for
fungi exhibited better resistance than the other species. The optimum pH for growth and accumulation of Cd was
observed to be 5. Uptake of Cd by adsorption was high for Aspergillus spp. compared to Bacillus spp. Per cent
growth reduction was high in Bacillus spp. than in Aspergillus spp. when exposed to Cd.
Keywords: Cadmium, Aspergillus, Bacillus, growing cells, biosorption
Introduction
Cadmium, a nonessential element to life forms is relatively accessible to biological systems and
is reported to be very toxic. Once the cadmium has been released into the environment, it exhibit
toxic effects on organisms and removing excesses from soil and water is deemed important for
environmental health. Since conventional wastewater treatments, such as chemical precipitation
or ion exchange and adsorption may be ineffective or uneconomical (Matheickal et al. 1997),
removal of cadmium using biosorption has been investigated in recent years. Microorganisms are
capable of actively and passively concentrating metals to levels substantially higher than those
found in their immediate environment. They have a high surface area-to-volume ratio because of
their small size and therefore provide a large contact area that can interact with metals in the
surrounding environment. The ability to accumulate and the capacity to grow in the presence of
relatively high concentrations of heavy metals depends on functional detoxification mechanisms.
Though different biomass types such as bacteria and fungi have been studied extensively in the
last decade, (Goksungur et al. 2005), the ability to resist and accumulate Cd by natural microbial
population in artificially spiked neutral soil have not been explored. Therefore, the study was
aimed to investigate the efficiency of living cells of three species of Bacillus, and Aspergillus
isolated from a sandy loam soil spiked with Cd to biosorb cadmium from aqueous solutions.
In view of the above, owing to the concept that metals come into contact with the
organisms via the liquid phase, in our present study, the effect of Cd on the growth and
accumulation in three different Bacillus species for bacteria and three different Aspergillus
species for fungi, isolated from Cd spiked soil (alfisol) was investigated in aqueous media, since
soils are very heterogeneous systems and thus complicated to study, and the properties of which
are modified continually by microbial, chemical, hydrological and geological processes. Such a
study would help in their application in wastewater treatment and the knowledge gained would
also help in understanding the mechanism by which they could retain the metals thereby reduce
their bioavailable levels in soils.
Materials and methods
Five hundred gram of uncontaminated soil was amended with 1000 μg g-1 Cd mixed well and
incubated for a period of 90 days at room temperature (28±2oC). Bacteria and fungi were isolated
using dilution plate technique and expressed as colony forming units per g of soil (CFU/g soil)
(Jha et al. 1992). The general characteristics of the soil are as follows: sandy loam in texture, bulk
density 1.4 g cc-1, pH 6.0 (1:2.5 soil water suspension), electrical conductivity 0.2 mS cm-1 and
organic carbon 0.4%. Soil available nutrient and metal concentrations in µgg-1 were; Cu=4.5,
Zn=12.5, Fe=5.4, Mn=31.1, N=600, P=10, K=39.0, Na=40.6, Ca=100, Mg=60, Cd=0.7 and
Se=0.2.
Selection, identification and storage of bacterial and fungal strains
Nutrient agar for bacteria and Czepek dox agar for fungi was prepared with different
concentrations of Cd in the range of 10, 50, 100, 200, 400, 800, 1000 and 1500 mg l -1. The
dominant isolates were streaked and the plates were incubated at room temperature for 24-48 h at
37oC for bacteria and at room temperature (27±2oC) for fungi for 4-5 days. Tolerant strains of
bacteria and fungi were identified and used in further comparative studies. The selected colonies
were purified by repeated streaking on nutrient agar for bacteria and Czepek Dox agar for fungi.
Bacterial identification was carried out at the Institute of Microbial Technology, Chandigarh,
India and fungal species were identified at Agharkar Research Institute, Pune, India. Bacterial
and fungal strains were maintained in agar slants containing nutrient and Czepek Dox broth,
respectively at 4oC. They were subcultured to maintain the culture purity.
Growth and accumulation experiments for Cd were carried in nutrient broth. Cadmium
solutions were prepared in sterile distilled water. For bacteria, the range of concentration of Cd
used was 0–60 mg l-1, and for fungal study, Cd concentrations ranged from 0–50 mg l-1.
Assessment of initial Cd concentration on the growth of bacteria and their accumulation
Growth and accumulation of Cd was carried out in 250 ml Erlenmeyer flasks containing 100 ml
of each concentration of heavy metal solution. One ml of pre harvested cultures from the
exponential growth phase was transferred to fresh medium amended with Cd solutions. Cell
density standardized at OD 600 nm ≈ 0.5 (Containing approximately 10 7 cells ml-1) was used.
Cells were allowed to grow in the metal amended media until the exponential phase (24 h).
Control was represented by the cultures in nutrient broth without metals. At the end of the
exponential phase, cells were harvested by centrifugation (4500 rpm, 10 min). Biomass dry
weight was determined by drying the pellets in an oven at 105 oC for 24 h (Costa and Duta,
2001). For fungus, about 15 agar plugs (Ø7 mm) containing the fungal colonies were
homogenized in liquid media and 1 ml of this was inoculated into 100 ml of Czepek dox broth
solution amended with the respective metal solutions. The cultures were incubated for 120 h at
160 rpm and at the end of the incubation period the fungal biomass were removed from
suspension through filtration, and the biomass dried in pre-dried, pre-weighed Whatman filter
paper No. 1 at 80oC until a constant weight was achieved.
Assessment of initial pH on the growth and accumulation of Cd
The effect of initial pH in the range of 4-8 on the uptake and accumulation of Cd by bacteria and
fungi at two different initial concentrations (10 and 40 mg l-1 Cd) was studied. To avoid
precipitation of metals at high pH, all experiments were carried out only up to pH 8. The initial
pH was adjusted with 1N NaOH and 1 N HNO3 as required. Growth and concentration of Cd
was estimated at the end of the experiment.
Extracellular and intracellular accumulation of Cd
The biomass of fungus grown in the presence of Cd was washed first with double distilled water
and then with 10 mM EDTA for 30 min. The bacterial cells were washed with distilled water and
were incubated for 15 min with an excess volume of ice-cold 5mM EDTA in 0.85% NaCl (pH
7.1) in order to remove the surface-bound metal (Anand et al. 2006). It was subsequently
centrifuged and the supernatant was collected for Cd estimation. The cell pellet was used for
intracellular metal estimation. Intracellular concentration of Cd was determined by digesting the
dried microbial biomass pellet in HNO3. The biomass was oven dried at 800C, and weighed. To
each pellet 5 ml of 6 M HNO3 was added and the mixture was left for 3 h. It was placed on hot
sand bath at 1200C to release cell associated metal ions. . The mixture was diluted with 25 ml
distilled water and estimated for Cd (Remacle, 1980). Cadmium was estimated using Graphite
Furnace Atomic Absorption Spectrophotometer (Varian Techtran Spectr AA 10/20 BQ). Per cent
adsorption was calculated using the following formula: [Adsorbed fraction / Total
bioaccumulated metal (Adsorption + Absorption)] x 100
Results
Selection of strains
Among bacteria Bacillus firmus and Bacillus simplex were able to tolerate and grow up to the
treatment concentration of 1500 µg g-1 of Cd in the agar medium. Among fungi, Aspergillus
niger was able to tolerate up to 1000 µg g-1 of Cd exposure. Cadmium caused a concentration
dependent decrease on the growth Bacillus simplex, Bacillus firmus and Bacillus ehimensis (Fig.
1). Cadmium also caused a concentration dependent growth decrease on the growth of
Aspergillus niger, Aspergillus wentii and Aspergillus glaucus (Fig. 2).
Effect of pH on accumulation
The growth and Cd accumulation of Bacillus and Aspergillus sp. exposed to different pH in the
range of 4 – 8, at initial Cd concentrations of 10 and 40 µg ml-1 showed significant differences.
For B. simplex at the initial Cd concentration of 10 µg ml-1, the maximum growth was recorded
at pH 7 and accumulation at pH 6. At 40 µg ml-1, maximum growth was at pH 6 (Fig. 3). B.
firmus recorded maximum growth and accumulation at pH 7, at the initial Cd concentration of 10
µg ml-1, whereas at 40 µg ml-1 Cd, maximum growth was at pH 6 and accumulation at pH 7.
Bacillus ehimensis recorded maximum growth and accumulation at pH 6, at 10 µg ml-1 Cd,
whereas at 40 µg ml-1 Cd, maximum growth was at pH 5 and accumulation at pH 6. A. niger
recorded the maximum growth and accumulation at pH 5, at both the concentrations of Cd.
At the initial Cd concentration of 10 µg ml-1, A. wentii recorded the maximum growth at
pH 7, and maximum accumulation at pH 5. At 40 µg ml-1 Cd, maximum growth and
accumulation was recorded at pH 5. Maximum growth and accumulation of A. glaucus at both
concentrations of Cd was at pH 5 (Fig. 4).
Intracellular accumulation and surface bound fraction by Bacillus Sp.
The intracellular accumulation of Cd by B. simplex increased with an increase Cd concentration
from 1 – 16 µg ml-1 of Cd. Further increase in Cd concentration from 18 to 60 µg ml-1 caused a
decrease in the accumulation of Cd, although the amount accumulated remained higher than that
of the control. Accumulation by Bacillus firmus increased with an increase in the treatment
concentration of Cd,. Accumulation by Bacillus ehimensis also increased with an increase in the
treatment concentration of Cd from 1 – 30 µg ml-1 of Cd. At 40 µg ml-1 treatment concentration
of Cd, it decreased, and with further increase in the treatment concentration significant changes
were not observed in the level of Cd accumulation (Fig. 5a-c). The surface bound fraction of Cd
by B. simplex recorded an increase with an increase in the Cd concentration from 1 – 16 µg ml-1.
When Cd concentration was increased, there was a decrease in the surface bound fraction of Cd,
however it was higher than the control. The surface bound fraction of Cd in cells of B. firmus
and B. ehemensis also increased with an increase Cd concentrations (Fig. 5 a-c).
Intracellular accumulation and surface bound fraction by Aspergillus Sp.
The intracellular accumulation of Cd by A. niger increased with increasing concentration of Cd.
A. wentii and A. glaucus also showed an increase in the intracellular accumulation of Cd. At the
highest treatment concentration (50 µg ml-1), growth inhibition was observed in A. wentii and A.
glaucus (6a-c). The surface bound fraction of Cd in cells of A. niger, increased with an increase
in Cd concentrations. (Fig. 6 a-c). The surface bound fraction of Cd in cells of A. wentii and A.
glaucus also increased with an increase in the treatment concentration of Cd, however, the
growth of the cells was inhibited at 50 µg ml-1 treatment concentration of Cd (Fig. 6 a-c)
Discussion
The variation recorded in the degree of effect of Cd on the three species of Bacillus and
Aspergillus may be due to variation in the type and degree of resistance mechanisms operating in
them against Cd. Among the three species of Aspergillus, A.niger exhibited better resistance
compared to A.glaucus and A.wentii. The increased growth rate recorded at 1 µg ml-1 in the case
of A. wentii may be due to a mechanism employed by it to overcome the shock of metal exposure
as has been reported by Moore et al. (2008), Duddridge and Wainwright, (1980) and Hassen et
al. (1998) in a heavy metal tolerant fungal strain. Although the cells showed reduced growth at
higher concentrations of Cd, the cells were alive, indicating their ability to tolerate the toxic
effect of Cd by diverting all the energy towards metal detoxification as have been reported by
Perez-Rama et al. (2002) and Malik (2004) with reference to Cd accumulation by Tetraselmis
suecica.
A decrease in growth was observed at lower and higher pH values indicating that the
optimum pH for growth was around 5-7 for Bacillus spp. The optimum pH for growth of
Aspergillus spp. was in the range 5-7. At lower pH of 3 and 4, in the presence of Cd A. niger
formed unusually large, smooth, round mycelial pellets, It resulted in branched distortions of the
hyphae. These morphological changes may have clumped the hyphal mat together, resulting in
the formation of the large round mycelial bodies. Such a morphological formation has been
reported for A. niger in the presence of nickel in acidic pH (Magyarosy et al. 2002).
Accumulation of Cd by Bacillus spp. and Aspergillus spp. revealed that optimum pH for
sorption was in the pH range 5-7 which correlated with the observed pH range for optimal
growth. The increase in accumulation of Cd with increase in pH from 4-7 may be because, as pH
increases, functional sites on the microbial surface become deprotonated and may bind cationic
metals. Accumulation starts to decrease again at pH>7, because microorganisms may secrete
soluble organic compounds which bind metals and thus act as “competitors”. Further more, the
microbial surface and its metal binding characteristics change in response to the increased pH. At
acidic pH, metal cations and protons (H+) compete for non-specific sorption sites on the biomass
thus making the number of positively charged (protonated) sites more abundant. This creates a
repulsive ionic environment resulting in reduced Cd uptake as has been reported for Pb and Cu
(Sekhar et al. 1998). As the pH increases from 4-7, more groups with overall negative charge
dominate; for example the carboxyl groups become deprotonated and are able to attract cations,
thus explaining higher Cd accumulation with increase in pH. The chemical state of the biomass
surface functional groups, which are responsible for metal binding, is also being influenced.
Therefore, it is possible that higher pH values can result in easier transportation of Cd cations
into the cell (cytosol). However, investigation of pH values above 8 was not feasible, since
metals precipitate (Klimmek et al. 2001). In the case of Cd the issue is more complicated since
microprecipitation occurred above pH 7 (Keffala et al. 1999) which may lead to erraneous
conclusion of sorption related removal from the medium.
Accumulation of cadmium by adsorption
Passive uptake of metals by microorganisms can be important since most heavy metals can be
adsorbed onto the surface of microbial cells. The amount of Cd adsorbed to the cell surface was
higher at higher concentrations of Cd, indicating the toxic effects of Cd on cells, which
consequently reduced accumulation of Cd within the cells. Bacillus spp. showed differential
uptake of Cd by adsorption, higher adsorption capacity exhibited by B. simplex.
Aspergillus niger showed high adsorbed Cd than intracellular uptake, ranging from 6.12
µg g at 0.5 µg ml-1 of Cd to 9389.24 µg -1 at the 50 µg ml-1 of Cd. Biosorption increased with
an increase in Cd concentration, although the biomass decreased. Even at the lowest Cd
concentrations, more than 50 % of the total uptake was by adsorption onto the cell surface. In A.
wentii, nearly 70 % of the total metal uptake was due to adsorption with 2338.26 µg g-1at 40 µg
ml-1, however the total Cd uptake computed was higher in A. niger. More than 50 % of Cd
uptake was by adsorption in A. wentii as was observed with A. niger. Comparatively, A. glaucus
recorded reduced Cd uptake by adsorption with adsorbed Cd content exceeding 50% of total Cd
uptake only at 10 µg ml-1 Cd, with Cd adsorbed ranging from 1.99 µg g-1 at 0.5 µg ml-1 to
1287.23 µg g-1 at 40 µg ml-1 Cd. Sorption of metals to the surface of cells is likely to play a
critical role in all microbe–metal interactions (Ledin, 2000).
-1
Bacteria possess cell wall or envelope that is capable of passively adsorbing high levels
of dissolved metals, usually via a charge mediated attraction (Macaskie and Dean, 1990).
Bacterial cell walls represent a large percentage of the total surface area exposed to fluids and
exhibit a strong tendency to adsorb aqueous metal cations (Konhauser et al. 1993). This may be
because of the possibility of complexation with polygalacturonic acid, an important constituent
of the outer layers of bacterial cells (Malik, 2004). In the case of fungus, surface binding of
cations is by binding to negatively charged groups on the hyphal surface, as was with the binding
of zinc in Neocosmospora vasinffecta (Platon and Budd, 1972) and binding of cobalt by
Neurospora crassa (Venkateswerlu and Sastry, 1970). The cell wall of fungi is the first to come
into contact with metal ions in solution, where the metals can be deposited on the surface or
within cell wall structure before interacting with the cytoplasmic material or other cellular parts
(Gadd, 1990). Chitin and chitosan contents of the fungal cell wall have been shown to sequester
metal ions (Tsezos, 1983). In fungal cells, a sophisticated network exists to buffer toxic
concentrations of heavy metals in their environment and to regulate intracellular concentrations
(Krauss et al. 2005; Clemens and Simm, 2003). Differences in rate of biosorption could be due to
the differences in cell wall structures between species (Gadd and Sayer, 2000, Miersch et al.
2001, Tobin, 2001). Higher biosorption could reflect a higher rate of bioprecipitation on the
mycelial surface (Breierova et al. 2002). In the Cd tolerant filamentous fungus Curvularia sp.
tolerance towards Cd is achieved by biosorption on the mycelial surface (Rama Rao et al. 1997
b). The cell walls of filamentous fungi appear to have the major role in biosorption due to
possession of numerous uptake sites. Higher uptake of Cd was observed at the lower biomass
concentrations, the lower uptake at high biomass concentrations, which can be attributed to the
electrostatic interactions of the functional groups at the cell surfaces. The cells at higher
concentrations in suspension attach to each other, thus lowering the cell surface area in contact
with the solution.
Intracellular accumulation
Lower metal concentrations did not result in significant uptake of metals by the Bacillus species.
Among the Bacillus spp. B. firmus showed high intracellular Cd of 7512.30 µg g-1 at 60 µg ml-1
Cd. Uptake by Bacillus simplex was 1875.42 µg g-1 at 16 µg ml-1Cd, and by B. ehemensis
1627.54 µg g-1 at 30 µg ml-1 Cd. The order of accumulation among the Bacillus spp. and
Aspergillus spp. was difficult to compare, since the trend of accumulation varied with increase in
Cd concentrations. However, the order of accumulation based on mean accumulation values was
B. firmus (1877 µg g-1) > B. simplex (756 µg g-1) > B. ehimensis (409 µg g-1). Among
Aspergillus spp., the order of accumulation was A. niger (354 µg g-1) > A.glaucus (255 µg g-1) >
A. wentii (224 µg g-1). During intracellular sequestration metals are accumulated in the
cytoplasm due to the presence of metallothioneins and cysteine-rich proteins, which bind metal
ions as has been reported by Silver et al. (1989). When the biomass concentration is low, metal
ions in the solution would not only be adsorbed to the surface of the biomass, but also enter into
intracellular part through facilitating the concentration gradient of metal ion. When the
extracellular concentration of metal ions was higher than that of intracellular, metal ions could
penetrate into the cell across the cell wall and membrane of the biomass by free diffusion. Metal
ions can also enter into the cell if the cell wall was disrupted by natural force (eg., autolysis) or
artificial force (mechanical force or alkali treatment etc). The above process is independent of
metabolism. Metal ions transported across the cell membrane, are transformed into other species
or precipitated within the cell by active cells, including transportation.
Living cells with active uptake system are more efficient in the sequestration of metals
since it was confirmed that polyphosphate bodies played a major role in accumulation of metals
(Torres et al. 1998). Uptake of Cu/Cr by bacterial strains has been reported in spite of their
growth and metabolism severely affected due to metal stress (Hassen et al. 1998). Suh et al.
(1998) has reported 10 times and 3 times higher accumulation capacity by live yeast cells
Saccharomyces cerevisiae and A. pullulans respectively than autoclaved cells. Bacillus circulans
EB1 cells grown in the presence of 28.1 µg ml-1 Cd were capable of removing Cd with a specific
biosorption capacity of 5.8 µg g-1 dry weight biomass (Yilmaz and Ensari, 2005). Efficient Zn
uptake by growing cells of Aspergillus spp., isolated from industrial waste has been reported
(Sharma et al. 2000; 2002). Higher Cd removing capacity of the living cells of the gram negative
bacteria S. paucimobilis than that of non living cells has been reported (Tangaromusk et al.
2002). Kapoor et al. (1999) reported a biphasic process of metal uptake in A. niger grown in
industrial gold mining solution. It has accumulated Au, Ag, Cu, Fe and Zn by precipitating them
on the cell surface (Gomes et al. 1999). Jaeckel et al. (2005b) has reported mycelial biosorption
and intracellular accumulation of Cd in Heliscus lugdunensis and Verticillum cf. alboatrum. Both
strains showed higher biosorption for Cd than intracellular accumulation of this metal, as has
been observed in the present investigation in A. niger. Studies of Perez-Rama et al. (2002)
demonstrated that although the algal cells (T. suecica) did not show growth at very high
concentrations of Cd (45 µg ml-1), the cells were alive and were synthesizing higher amounts of
sulfhydryl groups to tolerate the toxic effect of Cd. Under such conditions, cells can silently
continue accumulating Cd intracellularly without showing the apparent growth but diverting all
the energy towards metal detoxification. This facet of live and growing cells to metabolically
respond to high metal concentration has no counterpart in pure biosorption processes using dead
/ treated biomass.
Conclusions
Results of the present study suggest that growing cells of Bacillus firmus and Aspergillus niger
are potential candidates to grow in the presence of Cd and accumulate Cd thereby removing
cadmium from the solution efficiently. Such a removal efficiency of the organisms may render
them suitable for efficient metal removal by employing them in bioreactors for waste water
treatment contaminated with Cd.
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Figure 1. Effect of Cd on growth of Bacillus sp.
Weight (g)
0.25
0.2
0.15
0.1
0.05
Control
1
2
4
5
6
8
10
12
14
16
18
20
30
40
50
60
0
Cd, µg g-1
B. simlex
B. firmus
B. ehemensis
Weight (g)
Figure 2. Effect of Cd on growth of Aspergillus sp.
1.2
1
0.8
0.6
0.4
0.2
0
Cd, µg g-1
A. niger
A. wentii
A. glaucus
Figure 3. Effect of initial pH on the growth and accumulation of Bacillus sp.
1.2
1
0.8
0.6
0.4
0.2
0
1000
750
500
250
0
4 5 6 7 8 4 5 6 7 8 4 5 6 7 8
A. niger
A. wentii
A. glaucus
Cd accumulation, ug g-1
Weight (g)
Figure 4. Effect of initial pH on the growth and Cd accumulation by Aspergillus sp.
pH
Biomass
Cd accumulation
Figure 5a. Cd accumulation in B. simplex- Intracellular and surface bound
Accumln, µg g-1
2500
2000
1500
1000
500
Control
1
2
4
5
6
8
10
12
14
16
18
20
30
40
50
60
0
Cd, µg g-1
Intracellular
Adsorbed
Figure 5b. Cd accumulation in B. firmus - intracellular an surface bound
Accmn, µg g-1
15000
10000
5000
0
Cd, µg g-1
Intracellular
Adsorbed
Figure 5c. Cadmium accumulation in B. ehemensis - intracellular and surface bound
Accmn, µg g-1
2000
1600
1200
800
400
Control
1
2
4
5
6
8
10
12
14
16
18
20
30
40
50
60
0
Cd, µg g-1
Intracellular
Adsorbed
Figure 6a. Cd accumulation in A. niger - Intracellular and surface bound
10000
Accmln, µg g-1
7500
5000
2500
0
Cd, µg g-1
Intracellular
Adsorbed
Figure 6b. Cd accumulation in A. wentii - Intracellular and surface bound
Accmln, µg g-1
2500
2000
1500
1000
500
0
Cd, µg g-1
Intracellular
Adsorbed
Figure 6c. Cd accumulation in A. glaucus - Intracellular and surface bound
Accmln, µg g-1
1500
1000
500
0
Cd, µg g-1
Intracellular
Adsorbed