Potential of Live Biomass of Aspergillus Spp. in Biosorption of Heavy Metals from Aqueous Solutions

Full text

216 JOURNAL OF SOLID WASTE TECHNOLOGY AND MANAGEMENT VOLUME 43, NO. 3 AUGUST 2017
POTENTIAL OF LIVE BIOMASS OF ASPERGILLUS SPP. IN
BIOSORPTION OF HEAVY METALS FROM AQUEOUS SOLUTIONS
A. B. Gunjala, B. P. Kapadnisa*, N. J. Pawarb
aDepartment of Microbiology, Savitribai Phule Pune University, Ganeshkhind Road, Pune 411007
bDepartment of Geology, Savitribai Phule Pune University, Ganeshkhind Road, Pune 411007
INDIA
Tel: 020-25690643; Fax: 020-25690643
bpkapadnis@yahoo.com
ABSTRACT
Heavy metal pollution of soil, water and air is one of the major issues many countries are fac-
ing. The fungi were isolated from the compost and compost yard soil and identified as
Aspergillus clavatus, Aspergillus oryzae and Aspergillus fumigatus and studied for their ability
to sequester heavy metals from solution. The sequestration of Zn and Pb was more by A.
oryzae which was 2.96 and 9.93 mg g-1, respectively, Cd by A. fumigatus which was 19.24 mg
g-1 and Ni by A. clavatus which was 6.35 mg g-1 from the mixed metal solutions. Sequestration
of Cd, Pb and Ni from the mixed metal solutions was insignificant using the mixed biomass.
Sequestration of Zn was more by A. oryzae which was 24.73 mg g-1, Cd by A. fumigatus which
was 19.94 mg g-1, while Pb and Ni by A. clavatus which was 14.06 and 10.38 mg g-1,
respectively from the individual metal solutions. The sequestration of heavy metals was not ef-
fective from individual metal solutions using the mixed biomass. The optimum biomass was 0.5
g, PH and temperature were 5.0 and 38oC, respectively where more than 95% sequestration of
Zn, Cd and Ni was found by A. clavatus, A. oryzae and A. fumigatus respectively. The seques-
tration of heavy metals Zn, Pb and Ni mg g-1 by the fungal biomass increased with increase in
metal concentration. Biosorption of heavy metals will be uncomplicated, reusable and rapid for
control of heavy metal pollution.
Keywords: Heavy metal; Compost; Biosorption; Biological; Waste-treatment
INTRODUCTION
In recent years, heavy metal pollution has become one of
the most serious environmental problems. Presence of heavy
metals even in traces is toxic and detrimental to both flora
and fauna. With the rapid development of many industries
(mining, surface finishing, energy and fuel producing, ferti-
lizer, pesticide, metallurgy, iron and steel, electroplating,
electrolysis, electro-osmosis, leather, photography, electric
appliance manufacturing, metal surface treating) and aero-
space and atomic energy installations, wastes containing met-
als are directly or indirectly being discharged into the envi-
ronment causing serious environmental pollution and even
threatening human life (Volesky, 1990a; Wang, 2002a).
The impact of heavy metal release into our environment is
also increasing as a result of population explosion, haphazard
rapid urbanization, industrial and technological expansion,
increased energy utilization and waste generation from do-
mestic and industrial sources. These have rendered many
waters unwholesome and hazardous to man and other living
____________________________________
*Corresponding author
https://doi.org/10.5276/JSWTM.2017.216

POTENTIAL OF LIVE BIOMASS OF ASPERGILLUS SPP. IN BIOSORPTION OF HEAVY METALS FROM AQUEOUS SOLUTIONS 217
resources. The release of these heavy metals poses a signifi-
cant threat to the environment and public health because of
their toxicity, bioaccumulation in the food chain and persis-
tence in nature (Cerabasi and Yetis, 2001). The heavy metal,
Zinc (Zn) causes damage to the nervous system, Cadmium
(Cd) causes cancer, Lead (Pb) can cause mental retardation in
children and also damage kidney and liver and Nickel (Ni)
can cause stomach and intestinal irritation (Mudgal et al.,
2010). Therefore, it is very important to sequester the accu-
mulated heavy metals.
The conventional chemical methods for the sequestration
of heavy metals include precipitation, ion-exchange, electro-
chemical processes and membrane technology (Matheickal
and Yu, 1999). But all the chemical methods have proved to
be much costlier, time-consuming, non-reusable and less effi-
cient than the biosorption process (Volesky and Holan, 1995;
Amuda and Ibrahim, 2006; Kapoor et al., 1999; Pagnanelli et
al., 2001). In addition, chemical methods generate secondary
pollutants. “Biosorption” is a process in which solids of natu-
ral origin, such as microbial biomass (live or dead) or their
derivatives are employed for sequestration of heavy metals
from an aqueous environment. It has received substantial
attention as a potential method for decontamination and re-
covery of heavy metals from the environment (Lewis and Kiff
1988; Luef et al., 1991; Venkateswerlu and Stotzky, 1989).
Biosorption, a biological method of environmental control,
can be an alternative to conventional waste-treatment facili-
ties.
Fermentation industries all over the world generate huge
amounts of waste biomass, which are used in animal feed,
organic manure or incinerated. In a day, antibiotics fermenta-
tion industries generate about nearly 5000 tons of fungal bi-
omass which is very huge (Fourest and Roux, 1992; Paknikar
et al., 1993). The potential use of waste biomass in metal
removal remains untapped. However, such studies have re-
cently shown considerable interest among researchers.
Mycelial wastes of Rhizopus arrhizus (Fourest and Roux,
1992), Penicillium chrysogenum (Paknikar et al., 1993) and
Streptomyces pimprina have been studied extensively in the
toxic metal removal processes. Such an alternative is eco-
nomical and also attractive as disposal of fermentative waste
itself remains a serious problem. Also, biomaterials like fungi
have been proven more efficient and economical for the se-
questration of toxic metals from dilute aqueous solutions by
biosorption because of their filamentous morphology and cell
wall composition (Addour et al., 1999). Moreover, large
quantity of fungal biomass is available from the antibiotic,
food industries and biological waste management systems.
The use of microbial biomass to extract heavy metals
from effluents is an area of extensive research and develop-
ment (Scott and Palmer, 1990). Intact microbial cells, living
or dead, and derived microbial products, can be highly effi-
cient bioaccumulators of both soluble and particulate forms
of metals, especially from dilute solutions (Gadd and de
Rome, 1988). Earlier investigations have shown that
biosorption of heavy metals by microorganisms is a rapid and
reversible reaction that is not necessarily mediated by meta-
bolic processes (Shumate and Strandberg, 1985). Processes
using dead cell biomass can be of great interest because of
the large variety and low cost of these biological materials
(Fourest and Roux, 1992; Fourest et al., 1994).
Fungal biomass has been found to be excellent biosorbent
for heavy metals (Holan and Volesky, 1994; Volesky and
Holan, 1995). Fungal biomass has been used to sequester
copper, lead, zinc, nickel, cadmium, gold, silver and various
actinide elements, such as thorium, uranium and plutonium
(Gadd and White, 1989; Meyer and Wallis, 1997; Tsezos and
Volesky, 1981).
Fungi are known to have good metal uptake systems with
metabolism-independent biosorption being the most efficient.
Potential of filamentous fungi in bioremediation of heavy
metal containing industrial effluents and wastewaters has
been increasingly reported from different parts of the world
(Gadd, 1993). The specific mechanisms of uptake differ
quantitatively and qualitatively according to the species, the
origin of the biomass and its processing (Tobin et al., 1984)
and like other microorganisms can absorb heavy metals from
their external environment by means of physico-chemical and
biological mechanisms. The hyphal wall was found to be a
primary site of metal ion accumulation. This is attributed to
several chemical groups (the acetamido group of chitin, ami-
no and phosphate groups in nucleic acids, amino, amido,
sulfhydryl and carboxyl groups in proteins, and hydroxyls in
polysaccharides) that might attract and sequester metal ions
(Holan and Volesky, 1995). Biomass of fungi, such as
Absidia, Cunninhamella, Mucor and Rhizopus exhibit excel-
lent metal ion uptake (Mueler et al., 1992). This could be due
to the high chitin and chitosan content of the cell walls of
these fungi.
There are reports which show that distillery spentwash-
based composts are dominant with various fungi viz.,
Aspergillus, Penicillum, Fusarium, Acremonlum and
Cladosprium (Hultman et al., 2010).
The present study reports for the first time the use of live
fungal biomass of Aspergillus clavatus, Aspergillus oryzae,
Aspergillus fumigatus isolated from spent wash-based com-
posts and compost yard soil, to sequester heavy metals Zn,
Cd, Pb and Ni from the aqueous solution which will be of
highly industrial relevance for the environmental protection.
MATERIAL AND METHOD
Distillery spentwash-based compost
Compost is prepared at the sugar factories using the dis-
tillery spentwash. The raw materials used are Pressmud, Farm
Yard Manure (FYM), Bagasse, etc. The distillery spent-wash
based composts and compost yard soil were obtained from
the sugar factories viz., Theur, Malegaon and Baramati.
Isolation of fungi from distillery
spentwash-based composts and compost
yard soil
The fungi were isolated by the viable count method from
the distillery spentwash-based composts and compost yard

218 JOURNAL OF SOLID WASTE TECHNOLOGY AND MANAGEMENT VOLUME 43, NO. 3 AUGUST 2017
soil samples. The fungi isolated from the composts and com-
post yard soil samples were maintained on Potato dextrose
agar (PDA) containing (g l-1) [Cut potato in small pieces and
weighed 200 g, boiled in small amount of distilled water,
filtered and made up the volume to 1000 ml; dextrose, 20 and
agar, 30], Sabouraud dextrose agar [SDA g l-1: peptone,10;
dextrose, 40; agar, 30 and PH 6.0] and Czapex dox agar
[Cdox agar media g l-1: sucrose, 4; NaNO3, 0.20,
MgSO47H2O, 0.10; K2HPO4, 0.10; agar 30 and PH 6.8] slants.
Characterization of fungi by slide culture
technique
Half strength sterile Cdox agar medium was prepared.
Agar blocks (1 cm2) were cut and placed on sterile glass
slide. The fungal isolates were inoculated to the four corners
of the agar block. The slides were incubated at 28oC for three
days in the petriplate on the glass triangle kept on the filter
paper moistened with 20% glycerol. The staining was done
by lactophenol cotton blue containing [lactophenol cotton
blue g l-1: phenol crystals, 1000; lactic acid, 1000; glycerin,
2000 and methylene blue 7.5] and observed under Phase Con-
trast Microscope (Lawrence and Mayo, Pune) under 40X
lens.
Colony characters on different media
The three fungal isolates were spot inoculated on the
Cdox and malt extract agar media [malt extract agar media g
l-1: malt extract, 20; agar, 20 and PH 6.5] plates and incubated
at 28oC for 6 d (Thom and Raper, 1945).
Sequestration of heavy metals
Microbial biomass and media preparation. The fungi were
grown in sterile liquid Yeast Peptone Glucose (Guangyu and
Viraraghavan, 2000) (YPG) broth [YPG g l-1: yeast extract, 3;
peptone, 10; glucose, 20 and PH 4.5] in conical flasks on a
rotary shaker at 125 rpm at 28oC for 72 hours. The biomass
was harvested after 3 d and washed with generous amounts of
deionised water.
Biosorption of heavy metals. All the biosorption experiments
were conducted in separate solutions containing Zn2+, Cd2+,
Pb2+ and Ni2+ added in the form of Zn(NO3)2 (Qualigens,
Mumbai), Cd(NO3)2 (Sisco Research Laboratories, Mumbai),
Pb(NO3)2 and Ni(NO3)2 (Central Drug House Private Limited,
New Delhi), respectively.
Biosorption from mixed metal solutions
The biosorption was carried from the mixed metal solu-
tions using individual and mixed biomass. The biomass of
each fungus (2 g) was weighed and suspended individually
and in mixed form in mixture of heavy metal solutions of
1000 parts per million (ppm) of PH 5.0 (50 ml each) prepared
using the deionised water in 500 ml conical flasks and incu-
bated at 28oC under stationary conditions.
Biosorption from individual metal solutions. The biosorption
was carried from the individual metal solutions using indi-
vidual and mixed biomass following the previous procedures.
For above all of the biosorption experiments, after 20
minutes of contact time (Hany et al., 2004), the biomass was
filtered through Whatman No.1 filter paper and the filtrate
was analyzed for the metal content on Atomic Absorption
Spectrophotometer (AAS) (Varian SpectraA, Germany). The
amount of metal biosorbed g-1 of the biomass was calculated
using the following equation (Guangyu and Viraraghavan,
2000):
Q = [(Ci-Cf)/m]xV
where, Q is the metal ion bioadsorbed (mg g-1), Ci = initial
metal ion concentration (mg l-1)
Cf = final metal ion concentration (mg l-1),
m = biomass in the reaction mixture, (g)
V = volume of the reaction mixture (l)
Optimization of metal biosorption
parameters
Biomass concentration. The biomass of each fungus (0.5, 1.0
and 2.0 g) was weighed and suspended in 50 ml heavy metal
solution each of 1000 ppm of PH 5.0 in 500 ml conical flasks.
The flasks were kept for 20 minutes of contact time at 38oC,
filtered through the Whatman No.1 filter paper and the filtrate
was analyzed for the metal content.
PH. The biomass of each fungus (0.5 g) was suspended in 50
ml heavy metal solution of 1000 ppm of PH 3.0, 5.0 and 8.0,
respectively in 500 ml conical flasks. The flasks were kept
for 20 minutes of contact time at 38oC, filtered through the
Whatman No.1 filter paper and the filtrate was analyzed for
the metal content.
Temperature. The biomass of each fungus (0.5 g) was sus-
pended in 50 ml heavy metal solution of 1000 ppm of PH 5.0
in 500 ml conical flasks. The flasks were kept for 20 minutes
of contact time at temperatures 15, 28 and 38oC respectively,
filtered through Whatman No.1 filter paper and the filtrate
was analyzed for the metal content.
Metal concentration. The biomass of each fungus (0.5 g) was
suspended in 50 ml heavy metal solution of different concen-
trations viz., 5, 10, 15, 20 and 25 ppm of PH 5.0 in 500 ml
conical flasks. The flasks were kept for 20 minutes of contact
period time at 38oC, filtered through Whatman No.1 filter
paper and the filtrate was analyzed for the metal content.
RESULT AND DISCUSSION
Fungal population in distillery spentwash-
based composts and compost yard soil
Fungal population in distillery spentwash-based composts

POTENTIAL OF LIVE BIOMASS OF ASPERGILLUS SPP. IN BIOSORPTION OF HEAVY METALS FROM AQUEOUS SOLUTIONS 219
of Theur, Malegaon and Baramati was very high which was
21.3 x 104, 7.9 x 104 and 20.3 x 104 cfu g-1 respectively,
whereas in compost yard soil the fungal population was very
less which was 5.1 x 104 cfu g-1. This is because distillery
spentwash is rich in nutrients (mg l-1) like ammonical nitro-
gen (636.25), phosphorus (28.36), potassium (6500), calcium
(920), sodium (420) and metals (mg l-1) like Mg (753.25), Fe
(6.3), Mn (1429), Zn (1.09), Cu (0.265), Cr (0.067), Cd
(0.036) and Co (0.08) (Rath et al., 2010).
The fungal population on Sabouraud dextrose agar medi-
um was more as compared to that on Potato dextrose agar
medium. The fungal population in compost from distillery
spentwash of Theur, Malegaon and Baramati on Sabouraud
dextrose agar medium was found to be 24.60 x 104, 6.80 x
104 and 21.00 x 104 cfu g-1 respectively (Table 1), whereas
the fungal population in compost yard soil on Sabouraud dex-
trose agar medium was 3.73 x 104 cfu g-1 (Table 2).
It was also observed that fungal population was more in
the distillery spentwash-based composts of Theur and
Baramati (Table 1).
About 15 fungi were isolated from the distillery
spentwash-based composts and compost yard soil. These fun-
gi were studied for their lignocellulose degrading properties
and the most potential fungi were further selected for their
biosorption studies.
Characterization of the fungal isolates by
slide culture technique
Slide culture and staining by lactophenol cotton blue
showed the fungal species isolated from the compost and
compost yard soil samples were Aspergillus clavatus,
Aspergillus oryzae and Aspergillus fumigatus (Tsuneo, 2008).
 A. clavatus condiophores are erect, simple with
clavate conidial head. The condiophores are 125 µm
tall, vesicles 20 µm and phialides 6.5 x 2 µm in di-
ameter respectively. The conidia are 2.5 µm in di-
ameter.
 A. oryzae condiophores are hyaline, simple, 75 µm
tall, vesicles 40 µm and phialides 8.0 x 4.5 µm in
diameter respectively. The conidia are ellipsoidal
measuring 4.5-8.0 µm in diameter.
 A. fumigatus condiophores are hyaline, simple, in-
flated at the apex forming nodded vesicles bearing
conidial heads. The condiophores are 80 µm tall,
vesicles 16 µm and phialides 4.9 x 2.5 µm in diame-
ter respectively. The conidia are 2.7 µm in diameter.
Colony characters on different media
On Cdox solid agar media plates A. clavatus and A.
TABLE 1
Fungal population in distillery spentwash composts*
Medium
Fungal population (cfu g-1) x 104
A
B
C
PDA
18.0+0.00
9.0+1.47
19.7+1.20
SDA
24.6+0.00
6.8+0.80
21.0+3.00
cfu, colony forming units. Each data point represents average of triplicate + SD
*Distillery spentwash-based composts from 3 sources viz., Theur Malegaon and Baramati
TABLE 2
Fungal population in compost yard soil on different media
Medium
Fungal population (cfu g-1) x 104
PDA
5.80+0.90
SDA
3.73+0.35
Cdox
5.90+0.00
PDA, Potato dextrose agar; SDA, Sabouraud dextrose agar and Cdox, Czapex dox agar
Each data point represents average of triplicate + SD

220 JOURNAL OF SOLID WASTE TECHNOLOGY AND MANAGEMENT VOLUME 43, NO. 3 AUGUST 2017
fumigatus showed dark blue-green color colonies with diame-
ter 3.5 and 4.0 cm respectively, while A. oryzae showed ol-
ive-yellow colored colony with diameter 5.0 cm after incuba-
tion at 28oC for 6 d.
Colonies on the malt extract agar solid agar media grew
fast and showed similar growth characteristics.
Biosorption studies
Sequestration of metals from mixed metal solutions using
individual and mixed biomass . The sequestration of Zn and
Pb was more by A. oryzae which was 2.96+0.01 and
9.93+0.00 mg g-1 respectively from mixed metal solutions.
Cd sequestration was more by A. fumigatus which was
19.24+0.00 mg g-1, while Ni sequestration was more by A.
clavatus which was 6.35+0.01 mg g-1 as listed in Table 3.
The sequestration of Cd was 6.51+0.01 mg g-1 by A.
clavatus, A. oryzae and A. fumigatus. The sequestration of Pb
and Ni was very less from mixed metal solutions using mixed
biomass (Table 3).
There was direct competition for the binding sites
between Zn, Cd, Pb and Ni in the mixed metal solution
system where Cd uptake overtook the uptake of Zn, Pb and
Ni under the same conditions, i.e. metal concentration 1000
ppm, PH 5.0 and temperature 28oC. Zn biosorption by A.
clavatus, A. oryzae and A. fumigatus was significantly
affected due to presence of Cd, Pb and Ni metals because the
binding sites on the biosorbents were limited due to which the
metals competed simultaneously for the sites. There is a
report which has shown that Zn removal is reduced in
presence of cadmium in dual metal solution (Ting and Teo,
1994). The amount of suppression for Zn uptake depended on
the affinity of these ions for binding sites and binding
strength of the respective heavy metal ions to the biosorbent.
Sequestration of metals from individual metal solutions using
individual and mixed biomass. The sequestration of Zn was
more by A. oryzae which was 24.73+0.01 mg g-1, Cd
sequestration was more by A. fumigatus which was
19.94+0.00 mg g-1, while Pb and Ni sequestration was more
by A. clavatus which was 14.06+0.00 and 10.38+0.00 mg g-1
respectively from individual metal solutions as summarized
in Table 4.
The sequestration of heavy metals was not very effective
from individual metal solutions using mixed biomass (Table
4).
Studies have been done on biosorption of Pb (II) and Cd
(II) ions by the biomass of Phanerochaete chrysosporium
using single and binary metal solutions where the effects of
the presence of one metal ion on the biosorption of the other
metal ion were investigated in terms of equilibrium isotherm
and adsorption yield (Li et al., 2004). There is also a report on
adsorption study of Ag, Cu and Ni by the biomass of
Chrysosporium sp. using single and mixed metal solution
where the heavy metals when mixed in the water, the compe-
tition for adsorptive sites on the cell surfaces resulted in the
biomass adsorption of Ag being increased by 10 to 50%, but
Ni adsorption reduced by 10 to 80% and for Cu the adsorp-
tion reduced to 70% (Wu and Wang, 1995). Till date no work
has been on biosorption experiments carried out using mix-
ture of heavy metals and also no biosorption studies are done
using mixture of biomass of A. clavatus, A. oryzae and A.
fumigatus (Faryal et al., 2006; Iqbal et al., 2005).
Optimization of metal biosorption
parameters
Biomass concentration. 0.5 g of fungal biomass concentration
was found to be most favorable and effective where about
more than 95% sequestration of heavy metals Zn, Cd and Ni
was achieved by A. clavatus, A. oryzae and A. fumigatus as
listed in Table 5.
PH. The sequestration of heavy metals by A. clavatus, A.
TABLE 3
Sequestration of metals from mixed metal solutions by the biomass
Biomass
Sequestration (mg g-1) of
Zn
Cd
Pb
Ni
Individual biomass
A. clavatus
2.11+0.01
19.01+0.01
8.85+0.00
6.35+0.01
A. oryzae
2.96+0.01
17.21+0.01
9.93+0.00
0.28+0.01
A. fumigatus
0.21+0.00
19.24+0.00
6.40+0.01
6.13+0.00
Mixed biomass
A. clavatus, A. oryzae, A. fumigatus
-
6.51+0.01
2.81+0.00
0.12+0.01
Each data point represents average of triplicate + SD

POTENTIAL OF LIVE BIOMASS OF ASPERGILLUS SPP. IN BIOSORPTION OF HEAVY METALS FROM AQUEOUS SOLUTIONS 221
oryzae and A. fumigatus biomass showed that more than 95%
of Zn, Cd and Ni metal sequestration was achieved which
was very high (Table 6). This can be attributed to both sorp-
tion and surface precipitation of these metallic ions under
their free form. The sequestration of Zn, Cd and Ni at PH 5.0
by A. clavatus was 98.73, 99.73 and 95.73% respectively, by
A. oryzae 98.73, 99.45 and 95.76% respectively and by A.
fumigatus it was 98.73, 99.29 and 95.83% respectively (Table
6). Not much difference was observed in the percentage se-
questration of heavy metals with varied PH of heavy metal
solutions.
The results revealed that the effective and favorable PH
was 5.0 for the sequestration of heavy metals Zn, Cd and Ni
by A. clavatus, A. oryzae and A. fumigatus, which is similar
to the PH for biosorption of Zn by A. flavus RH07 and A.
fumigatus RH05 (Faryal et al., 2006). A. flavus RH07 showed
82.38% and A. fumigatus RH05 86.16% sequestration of Zn
at PH 5.0 (Faryal et al., 2006), whereas A. clavatus, A. oryzae
and A. fumigatus biomass showed 98.73% sequestration of
Zn at PH 5.0 from the aqueous solution which was high as
summarized in Table 6. It has been reported that optimal up-
take of Pb by Rhizopus arrhizus was achieved at PH 5.0
(Paknikar et al., 1993) which is similar to our result. Study
has been done where sequestration of Zn by Cunninghamella
echinulata was reported to be 89% at PH 5.0 (EI-Sayed and
EI-Morsy, 2004).
It has been reported that PH has important role in metal
ion biosorption, where the active biosorbing groups have the
ability to accept or loss of protons that depends mainly on the
PH value (Pinghe et al., 1999; Yalcinkaya et al., 2002).
Temperature. The sequestration of heavy metals Zn, Cd and
Ni by the fungal biomass was very high which was more than
95% at 15, 28 and 38oC temperature respectively (Table 7).
The % sequestration of Zn, Cd and Ni by A. clavatus, A.
oryzae and A. fumigatus was same at PH 5.0 and 38oC (see
Tables 6-7). Not much difference was observed in the per-
centage sequestration of heavy metals with varied tempera-
ture. Higher percentage sequestration of heavy metals from
15-38oC may be due to either high affinity of the binding
sites for the Zn, Cd and Ni cations or due to more availability
of the binding sites on the relevant cell mass of A. clavatus,
A. oryzae and A. fumigatus. The most effective temperature
was found to be 38oC for the sequestration of heavy metals
by A. clavatus, A. oryzae and A. fumigatus (Table 7).
At 28oC, non-living biomass of A. flavus RH07 and A.
TABLE 4
Sequestration of metals from individual metal solutions by the biomass
Biomass
Sequestration (mg g-1) of
Zn
Cd
Pb
Ni
Individual biomass
A. clavatus
0.10+0.00
16.92+0.00
14.06+0.00
10.38+0.00
A. oryzae
24.73+0.01
7.81+0.00
12.57+0.00
9.37+0.00
A. fumigatus
24.64+0.00
19.94+0.00
11.54+0.00
9.84+0.00
Mixed biomass
A. clavatus, A. oryzae,
A. fumigatus
-
-
1.17+0.00
5.75+0.00
Each data point represents average of triplicate + SD
TABLE 5
Sequestration of metals (%) by the biomass at varied concentrations
Biomass (g)
0.5
1.0
2.0
Zn
Cd
Ni
Zn
Cd
Ni
Zn
Cd
Ni
A. clavatus
98.73
99.73
95.73
98.04
98.90
93.02
97.92
98.48
92.80
A. oryzae
98.73
99.45
95.76
97.86
99.14
94.60
97.68
98.96
94.32
A. fumigatus
98.73
99.29
95.83
97.96
98.76
94.72
97.44
98.56
94.56
Each data point represents average of triplicate + SD

222 JOURNAL OF SOLID WASTE TECHNOLOGY AND MANAGEMENT VOLUME 43, NO. 3 AUGUST 2017
fumigatus RH05 showed 59.24% and 63.84% sequestration
of Zn after 30 min incubation. A. fumigatus at 28oC has
shown 98.73% sequestration of Zn which was very high after
20 min incubation (Table 7).
Metal concentration. As the metal concentration was in-
creased, it was observed that the sequestration of the heavy
metals Zn, Pb and Ni mg g-1 also increased by the fungal bi-
TABLE 6
Sequestration of metals (%) by the biomass at varied PH
PH
Biomass
3.0
5.0
8.0
Zn
Cd
Ni
Zn
Cd
Ni
Zn
Cd
Ni
A. clavatus
98.30
99.40
93.82
98.73
99.73
95.73
98.01
99.20
93.04
A. oryzae
98.50
99.21
95.34
98.73
99.45
95.76
98.01
98.90
94.72
A. fumigatus
98.42
99.02
95.70
98.73
99.29
95.83
98.26
98.90
94.90
Each data point represents average of triplicate
TABLE 7
Sequestration of metals (%) by the biomass at varied temperature
Temp (oC)
Biomass
15
28
38
Zn
Cd
Ni
Zn
Cd
Ni
Zn
Cd
Ni
A. clavatus
98.73
99.99
95.55
98.73
99.28
95.74
98.73
99.73
95.73
A. oryzae
98.74
99.11
95.69
98.73
99.15
95.76
98.73
99.45
95.76
A.fumigatus
98.73
99.12
95.68
98.73
99.37
95.79
98.73
99.29
95.83
Each data point represents average of triplicate
FIGURE 1
Sequestration of Zn by biomass at varied concentrations

POTENTIAL OF LIVE BIOMASS OF ASPERGILLUS SPP. IN BIOSORPTION OF HEAVY METALS FROM AQUEOUS SOLUTIONS 223
omass (Figures 1-3). This increase can be due to the increase
in electrostatic interactions, involving sites of progressively
lower affinity for metal ions, thus leaving more metal ions
unadsorbed in solution at higher concentration levels (Iqbal
and Edyvean, 2004). This trend was also for the removal of
lead and copper by Phanerochaete chrysosporium (Iqbal and
Edyvean, 2004). However, in case of Pb, as the metal concen-
tration was increased, the ability to sequester Pb by A.
fumigatus was decreased significantly (Figure 2). This sug-
gests that Pb metal-binding sites of A. fumigatus has got satu-
rated. Thus, biosorption increased with increase in concentra-
tion of the metals as long as the binding sites were available.
The sequestration of Zn by A. clavatus, A. oryzae and A.
fumigatus was high at 20 ppm which was 72% respectively
(Figure 1). The sequestration of Pb was found very less as
compared to the sequestration of Zn and Ni with varied metal
concentration.
There is a report on sequestration of copper ions by A.
oryzae from aqueous solution (Huang and Huang, 1996) and
also on chromium bioremoval from tannery industries efflu-
ent (Sepher et al., 2005). Studies have been done on
biosorption of Zn, Pb and Cu by Cunninghamella echinulata
where the effect of biomass concentration, PH and time of
contact were investigated (EI-Sayed and EI-Morsy, 2004).
Biosorption of Cd by dried biomass of A. fumigatus has been
studied where effect of PH, contact time, biomass concentra-
FIGURE 2
Sequestration of Pb by biomass at varied concentrations
FIGURE 3
Sequestration of Ni by biomass at varied concentrations
Each data point represents average of triplicate.

224 JOURNAL OF SOLID WASTE TECHNOLOGY AND MANAGEMENT VOLUME 43, NO. 3 AUGUST 2017
tion has been reported (Saleh Al-Garni et al., 2009). There is
also a report on biosorption potency of A. niger isolated from
soil and effluent of leather tanning mills in the sequestration
of chromium (VI) (Srivastava and Thakur, 2006). However,
till date there are not many reports found on biosorption of
Zn, Cd, Pb and Ni by A. clavatus, A. oryzae and A. fumigatus
where the effect of biomass concentration, PH, temperature
and different metal concentration has been studied.
CONCLUSION
The fungi, A. clavatus, A. oryzae and A. fumigatus have
been reported for the first time for the sequestration of heavy
metals Zn, Cd, Pb and Ni from the aqueous solutions. Also
the sequestration of heavy metals from the mixed and indi-
vidual metal solutions using the individual and mixed bio-
mass of A. clavatus, A. oryzae and A. fumigatus is reported
for the first time. About more than 95% sequestration of the
heavy metals was observed by A. clavatus, A. oryzae and A.
fumigatus. A technology can be developed for the sequestra-
tion of heavy metals from the individual metal solutions us-
ing the individual biomass. This will prove a very important
necessary application in the bioremediation processes for the
control of heavy metal pollution and will also be of high in-
dustrial relevance for the environmental protection.
The work demonstrates value-addition to the large amount
of biomass or sludge developed using various biological pro-
cesses. In comparison to all the processes viz., precipitation,
ion-exchange, electrochemical and membrane technology, the
biosorption technology adopted in this paper to sequester the
heavy metals will be very economical, reusable, rapid and
also the fungal biosorbent will be readily available in huge
amount.
ACKNOWLEDGEMENT
The authors are thankful to the University Grants Com-
mission (UGC), New Delhi, India for the financial support
for this research work.
REFERENCES
Addour, L., D. Belhocine, N. Boudries, Y. Comeau, and A.
Pauss, 1999. “Zinc uptake by Streptomyces rimosus bio-
mass using a packed-bed column.” J. Chem. Technol.
Biotechnol., Volume 74, pp. 1089-1095.
Amuda, O.S., and A.O. Ibrahim, 2006. “Industrial wastewater
treatment using natural material as adsorbent.” Afr J.
Biotechnol., Volume 5, pp. 1483-1487.
Cerabasi, I.H., and U. Yetis, 2001. “Biosorption of Ni (ii) and
Pb (ii) by Phanerochaete chrysosporium from binary met-
al system- Kinetics.” Water SA, Volume 27, pp. 15-20.
EI-Sayed, M., and EI-Morsy, 2004. “Cunninghamella
echinulata a new biosorbent of metal ions from polluted
water in Egypt.” Mycologia, Volume 96, pp. 1183-1189.
Faryal, R., A. Lodhi, and A. Hameed, 2006. “Isolation, char-
acterization and biosorption of zinc by indigenous fungal
strains Aspergillus fumigatus RH05 and Aspergillus flavus
RH07.” Pak J. Bot., Volume 38, pp. 817-832.
Fourest, E., and J.C. Roux, 1992. “Heavy metal biosorption
by fungal mycelial by-products: Mechanisms and influ-
ence of PH.” Appl. Microbiol. Biotechnol., Volume 37, pp.
399-403.
Fourest, E., C. Canal, and J.C. Roux, 1994. “Improvement of
heavy metal biosorption by mycelial dead biomass
(Rhizopus arrhizus, Mucor miehei and Penicillium
chrysogenum): PH control and cation activation.” FEMS
Microbiol. Rev., Volume 14, pp. 325-332.
Gadd, G.M., 1993. “Interactions of fungi with toxic metals.”
New Phytologist, Volume 124, pp. 25-60.
Gadd, G.M., and L. de Rome, 1988. “Biosorption of copper
by fungal melanin.” Appl. Microbiol. Biotechnol., Volume
29, pp. 610-617.
Gadd, G.M., and C. White, 1989. “Removal of thorium from
simulated acid process streams by fungal biomass.”
Biotechnol. Bioeng., Volume 33, pp. 592-597.
Guangyu, Y., and T. Viraraghavan, 2000. “Effect of pre-
treatment on the bioadsorption of heavy metals on Mucor
rouxii.” Water SA, Volume 26, pp. 119-123.
Hany, H., F.I. Soha, K. Kamal, and M. Hassan, 2004.
“Biosorption of heavy metals from wastewater using
Pseudomonas sp.” Electron. J. Biotechnol., Volume 7, pp.
38-46.
Holan, Z.R., and B. Volesky, 1994. “Biosorption of Pb and
Ni by biomass of marine algae.” Biotechnol. Bioeng.,
Volume 43, pp. 1001-1009.
Holan, Z.R., and B. Volesky, 1995. “Accumulation of cadmi-
um, lead and nickel by fungal and wood biosorbents.”
Appl. Biochem. Biotechnol., Volume 53, pp. 133-146.
Huang, C., and C.P. Huang, 1996. “Application of
Aspergillus oryzae and Rhizopus oryzae for Cu (II) re-
moval.” Water Res., Volume 30, pp. 1985-1990.
Hultman, J., T. Vasara, P. Partanen, J. Kurola, M.H. Kontro,
L. Paulin, P. Auvinen, and Romantschuk. 2010. “Deter-
mination of fungal succession during municipal solid
waste composting using a cloning-based analysis.” Appl.
Microbiol. Volume 108, pp. 472-487.
Iqbal, A, Z. Shaheen, and A. Farah, 2005. “Heavy metal
biosorption of Aspergillus and Rhizopus sp. isolated from
wastewater treated soil.” J. Appl. Sci. Environ. Manag.,
Volume 9, pp. 123-126.
Iqbal, M., and R.J. Edyvean, 2004. “Biosorption of lead, cop-
per and zinc ions on loofa sponge immobilized biomass of
Phanerochaete chrysosporium.” Miner. Eng., Volume 17,
pp. 217-223.
Kapoor, A., T. Viraraghavan, and D.R. Cullimore, 1999.
“Removal of heavy metals using the fungus Aspergillus
niger.” Biores. Technol., Volume 70, pp. 95-104.
Lewis, D., and R.G. Kiff, 1988. “The removal of heavy met-
als from aqueous effluents by immobilized fungal bio-
mass.” Environ. Technol. Lett., Volume 9, pp. 991-998.
Li, Q., S. Wu, G. Liu, X. Liao, X. Deng, D. Sun, Y. Hu, and
Y. Huang, 2004. “Simultaneous biosorption of cadmium

POTENTIAL OF LIVE BIOMASS OF ASPERGILLUS SPP. IN BIOSORPTION OF HEAVY METALS FROM AQUEOUS SOLUTIONS 225
(II) and lead (II) ions by pretreated biomass of
Phanerochaete chrysosporium.” Sep. Purif. Technol.,
Volume 34, pp. 135-142.
Luef, E., T. Prey, and C.P. Hubicek, 1991. “Biosorption of
zinc by fungal mycelial wastes.” Appl. Microbiol.
Biotechnol., Volume 34, pp. 688-692.
Matheickal, J.T., and Q. Yu., 1999. “Biosorption of lead (II)
and copper (II) from aqueous solution by pretreated bio-
mass of Australian marine algae.” Biores. Technol., Vol-
ume 69, pp. 223-229.
Meyer, A., and F.M. Wallis, 1997. “The use of Aspergillus
niger (strain 4) biomass for lead uptake from aqueous sys-
tems.” Water SA, Volume 23, pp. 187-192.
Mudgal, V., N. Madaan, A. Mudgal, R.B. Singh, and S.
Mishra, 2010. “Effect of toxic metals on human health.”
The Open Nutraceuticals J., Volume 3, pp. 94-99.
Mueler, M.D., D.C. Wolf, T.J. Beveridge, and G.W. Bailey,
1992. “Sorption of heavy metals by the soil fungi
Aspergillus niger and Mucor rouxii.” Soil Biol. Biochem.,
Volume 24, pp. 129-135.
Pagnanelli, F., M. Trifoni, F. Beolchini, A. Esposito, L. Toro,
and F. Vegli, 2001. “Equlibrium biosorption studies in
single and multi-metal systems.” Process Biochem., Vol-
ume 37, pp. 115-124.
Paknikar, K.M., U.S. Palnitkar, and P.R. Puranik, 1993.
“Biosorption of metals from solution by mycelial waste of
Penicillium chrysogenum.” In: Torma AE, et al., editors.
Biohydrometallurgical Technologies, vol II. Wyoming, USA:
The Minerals, Metals and Materials Society, TMS Pub-
lishers. pp. 229-235.
Pinghe, Y, Y. Qiming, J. Bo, and L. Zhao, 1999.
“Biosorption removal of cadmium from aqueous solution
by using pretreated fungal biomass cultured from starch
wastewater.” Water Res., Volume 33, pp. 1960-1963.
Rath, P., G. Pradhan, and M.K. Mishra, 2010. “Effect of sug-
ar factory distillery spentwash (DSW) on the growth pat-
tern of sugarcane (Saccharum officinarum) crop.” J. Phy-
tology, Volume 2, pp. 33-39.
Saleh Al-Garni, M, M.G. Khaled, and S.B. Abdulaziz, 2009.
“Biosorption characteristics of Aspergillus fumigatus in
removal of cadmium from an aqueous solution.” Afr. J.
Biotechnol., Volume 8, pp. 4163-4172.
Scott, J.A., and S.J. Palmer, 1990. “Sites of cadmium uptake
in bacteria used for biosorption.” Appl. Microbiol.
Biotechnol., Volume 33, pp. 221-225.
Sepher, M.N., S. Nasseri, M.M. Assadi, and K. Yaghmaian,
2005. “Chromium bioremoval from tannery industries ef-
fluent by Aspergillus oryzae.” Iran J. Environ. Health Sci.
Eng., Volume 2, pp. 273-279.
Shumate, S.E., and G.W. Strandberg, 1985. “Accumulation
of metals by microbial cells.” In: Moo-Young M, editor.
Comprehensive Biotechnology, vol IV. New York:
Pergamon Press. pp. 235-247.
Srivastava, S, and I.S. Thakur, 2006. “Biosorption potency of
Aspergillus niger for removal of chromium (VI).” Curr.
Microbiol., Volume 53, pp. 232-237.
Thom, C, and Raper, K.B., 1945. “A manual of the
Aspergilli.” Baltimore, Maryland: The Williams and Wil-
kins company Publishers. pp. 81-273.
Ting, Y.P., and W.K. Teo, 1994. “Uptake of cadmium and
zinc by yeast: Effects of co-metal ion and physi-
cal/chemical treatments.” Biores. Technol., Volume 50,
pp. 113-117.
Tobin, J.M., D.G. Copper, and R. J. Neufeld, 1984. “Uptake
of metal ions by Rhizopus arrhizus.” Appl. Environ.
Microbiol., Volume 47, pp. 821-824.
Tsezos, M., and B. Volesky, 1981. “Biosorption of uranium
and thorium. Biotechnol. Bioeng., Volume 23, pp. 583-
604.
Tsuneo, W., 2008. “Diversities of fungi.” In: Pictorial Atlas
of soil and seed fungi: morphologies of cultured fungi and
key to species, 3rd edn. Boca Raton, Florida: CRC Press.
Venkateswerlu, G., and G. Stotzky, 1989. “Binding of metals
by the cell walls of Cunninghamella blakesleeana grow-
ing in the presence of copper or cobalt.” Appl. Microbiol.
Biotechnol., Volume 31, pp. 619-625.
Volesky, B., 1990a. “Biosorption by fungal biomass.” In:
Volesky B, editor. Biosorption of heavy metals. Boca Ra-
ton, Florida: CRC Press. pp. 139-172.
Volesky, B., and Z.R. Holan, 1995. “Biosorption of heavy
metals.” Biotechnol. Prog., Volume 11, pp. 235-250.
Wang, J.L., 2002a, Immobilization techniques for biocata-
lysts and water pollution control. Beijing: Science Press.
pp. 233-248.
Wu, C.Y., and H.H. Wang, 1995. “The adsorption of silver,
copper and nickel by the biomass of Chrysosporium sp.”
J. Chin. Agric. Chem. Soc., Volume 33, pp. 30-36.
Yalcinkaya, Y., M.Y. Arica, L. Soysal, A. Denizli, O. Genc,
et al., 2002. “Cadmium and mercury uptake by immobi-
lized Pleurotus sapidus.” Turk. J. Chem., Volume 26, pp.
441-452.