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Submitted 30 October 2013, Accepted 17 December 2013, Published online 31 December 2013 1118
Corresponding Author: Marcelo A. Sulzbacher – e-mail – marcelo_sulzbacher@yahoo.com.br
Mycosphere 4 (6): 1118–1131 (2013) ISSN 2077 7019
www.mycosphere.org Article Mycosphere
Copyright © 2013 Online Edition
Doi 10.5943/mycosphere/4/6/9
Mucor racemosus as a biosorbent of metal ions from polluted water
in Northern Delta of Egypt
El-Morsy EM, Nour El-Dein MM and El-Didamoney SMM
Department of Botany, Faculty of Science, Damietta University, New Damietta, Egypt.
El-Morsy EM, Nour El-Dein MM, El-Didamoney SMM 2013 – Mucor racemosus as a biosorbent
of metal ions from polluted water in Northern Delta of Egypt. Mycosphere 4(6), 1118–1131, Doi
10.5943/mycosphere/4/6/9
Abstract
Twenty samples of polluted water were collected from Damietta's canals and drainages
located near the industrial area of New Damietta. Initial concentrations of heavy metals including
(zinc, copper and lead) in the polluted water were determined. Fourty–five fungal species were
isolated. Mucor racemosus, Aspergillus flavus, A. niger, A. fumigatus, Trichoderma koningi and
Rhizopus oryzae were isolated frequently. On the basis of its frequency, Mucor racemosus was
chosen for biosorption studies.
Free and immobilized biomass of Mucor racemosus sequestered ions in this decreasing
sequence Cu > Zn > Pb. The effects of biomass concentration, pH and time of contact were
investigated. The level of ion uptake rose with increasing biomass till 200 mg and then decreased
with increasing biomass. The maximum uptake for Cu (60.13 mg/g), Zn (57.67 mg/g) and Pb
(21.97 mg/g) respectively occurred at 200 mg/l biomass. The uptake rose with increasing pH up to
5 in the case of Zn and Cu and 4 in the case of Pb. Maximum uptake for all metals was achieved
after 15 minutes. Ion uptake followed the Langmuir adsorption model, permitting the calculation of
maximum uptake and affinity coefficients. Treatment of Mucor racemosus biomass with 0.1 M
NaOH at 120°C for 6 h improved biosorbent capacity, as did immobilization with alginate.
Immobilized biomass could be regenerated readily with treatment with dilute HCl. The biomass-
alginate complex efficiently removed Zn, Cu and Pb from polluted water samples. Therefore,
Mucor racemosus could be employed either in free or immobilized form as a biosorbent of metal
ions in waste water.
Key words – alginate – alkali treatment – biosorption – copper – free and immobilized biomass –
lead – zinc.
Introduction
The increasing trends towards artificial high life standards are compelling the people towards
misuse of resources resulting in environmental degradation at massive scale. Incidentally, increased
industrialization has affected the environment through disposal of waste water containing toxic
contaminants in the form of metals. This situation is getting more alarming in the last two decades
where industrial units are established without environmental impacts assessment and planning.
Therefore discharge of heavy metals, their accumulation and contamination has become an
environmental, health, economic and planning issue.
1119
Biological methods of metals removal from aqueous solution, known as biosorption have
been recommended as cheaper and more effective technique in bioremoval of heavy metals (Apel
&Torma 1993, Artola et al. 1997). Biosorption of metals is a property of certain types of microbial
biomass that can result in the concentration of metallic elements from relatively diluted solutions
(Volesky & Philips 1995). Biosorption has received substantial attention as a potential method for
decontamination and recovery of heavy metals from the environment (Lewis & Kiff 1988,
Venkateswerlu & Stotzky 1989, Luef et al. 1991). Fungal biomass and seaweed biomass have been
found to be excellent biosorbents for sequestering heavy metals (Lewis & Kiff 1988, Holan &
Volesky 1994, Volesky & 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 (Tsezos & Volesky 1981, Gadd & White 1989, Luef et al. 1991, Kapoor &Viraraghavan
1998a & b).
Fungi are known to have good metal uptake systems (Gadd 1986) with metabolism-
independent biosorption being the most efficient. The specific mechanism of uptake differs
quantitatively and qualitatively according to the species, the origin of the biomass and its
processing (Tobin et al. 1984). 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, amino
and phosphate groups in nucleic acids, amino acids, amido, sulfhydryl and carboxyl groups in
proteins and hydroxyls in polysaccharides) that might attract and sequester metal ions (Holan &
Volesky 1995).
Biomass of fungi such as Absidia, Cunninghamella, Mucor and Rhizopus, exhibit excellent
metal-ion uptake (Venkateswerlu & Stotzky 1989, Luef et al. 1991, Fourest & Roux 1992, Mueler
et al. 1992). This could be due to the high chitin and chitosan content of the cell walls of these
fungi (Tsezos & Volesky 1981). To date, research in the area of biosorption suggests it to be an
ideal alternative for decontamination of metal containing effluents (Yazdani et al. 2010, Nur Liyana
et al. 2011, Nitin et al. 2012, Siddiquee et al. 2013). Generally speaking the biosorption takes place
by both living and non-living microbial biomass, but there are differences in the efficiency and
mechanisms involved (Park et al. 2005). The efficiency by which dead cells act as sorbent of metal
ion may be greater than that of living cells and confirm its wider acceptability. This is due to the
argument that dead cells do not have toxicity limitations, no requirement of growth and nutrient
media, storage property for extended time period and easy desorption of adsorbed metal ions
(Awofolu et al. 2006).
Various physical (heat treatment, autoclaving, freeze drying and boiling) and chemical (acids,
alkali and organic chemicals) pretreatment protocols have been developed to convert the viable
cells into nonviable (dead) (Loukidou et al. 2003). According to many workers, the chemical
pretreatment protocols owing to change in cell wall chemistry of the biosorbent (Kapoor et al.
1999). Yan & Viraraghavan 2000 has reported that alkaline (caustic) treatment could enhance metal
binding by biomass.
The alkaline treatments, including sodium hydroxide, potassium hydroxide, alkaline
detergents or other alkaline reagents ruptures the cell walls of microbes and exposes additional
functional groups for metal ion binding. The residual alkalinity may result in the hydrolysis of
certain metals, thus enhancing the biosorption capacity of biomass (Brierley 1990). According to
Huang (1996) acid-washing process have better results over other treatments as this treatment may
dissolve polysaccharide components in the outer cell wall layer of the biosorbent, thus producing
additional binding sites. It is obvious that many different and challenging contributions have been
made on the path to develop biosorption by the biosorbent.
Therefore the aim of this study was to isolate fungi from polluted water to find a new
biosorbent agent. The ability of free and immobilized biomass of this biosorbents to sequester zinc
(Zn), copper (Cu) and lead (Pb) was investigated.
1120
Materials and Methods
Sampling procedures and isolation methods
Twenty water samples were collected in May 2010 from polluted water of Damietta's
drainages and canals located near the industrial area of New Damietta in clean, sterilized 1 L screw-
cap glass bottles. To each sample, 372 mg/l of EDTA (ethylene diamine-tetraacetic acid disodium
salt) was added as a chelating agent to reduce toxicity in samples laden with heavy metals. Other
twenty samples were also collected in clean, sterilized screw-cap glass bottles for the determination
of heavy metals concentration in the polluted water samples.
Examination of samples began immediately after return to the laboratory. For isolation of
fungi a millipore filter paper (0.45 mµ pore size) technique was used to concentrate fungal
propagules from the polluted water. The fungal propagules were resuspended in sterilized bottles
containing 50 or 100 ml of sterilized water, and 1 ml from each dilution was transferred to a clean
sterilized petri-dish (three replicates) contained streptopenicid (0.35g/ml ), rose bengal
(0.035gm/ml) glucose (10 gm/l), peptone (5gm/l) and agar (15gm/l) (Cooke 1963). The plates were
incubated at 28ºC for 4–7 days. The relative frequency of occurrence was calculated as the number
of species isolated from each sample divided by the total number of samples. The isolated species
were classified as very frequent (>20%), frequent (10-20%), or infrequent (<10%) as adapted from
(Tan and Leong 1989). Sporulating isolates were identified using specific media that were used for
identifying sporulating isolates. Non-sporulating strains were grouped as mycelia sterilia according
to similarities in colony morphology (Taylor et al. 1999).
Metal biosorption by free fungal biomass
Mucor racemosus was selected based on its frequent occurrence in sampling. The fungus
was subcultured on Potato Dextrose broth at 28ºC for 7 days on a rotatory shaker at 180 rpm.
Fungal pellets were washed twice in sterile double distilled water, drained and dried at 60ºC to
constant weight and ground with a mortar and pestle before determination of metal biosorption (El-
Morsy 2004).
Effect of initial metal concentration
To evaluate the effect of initial metal ion concentration (ci) on adsorption behaviour of Zn,
Cu and Pb by dried mycelial biomass, aliquots of 10, 50, 70, 100, 150, 200,300, 400 and 600 ppm
concentration of zinc sulfate, copper sulfate and lead acetate solutions were added to 250 ml
Erlenmeyer flasks with a fixed biomass of 200 mg/l. The pH was adjusted to 4 with 0.1N HCl and
0.1N NaOH. The samples were mixed well by shaking. For sorption isotherm experiments, flasks
were agitated on a rotatory shaker (180 rpm) at room temperature until no additional metal was
removed (3-5 h). The samples were filtered through 0.45 mµ millipore filters. Triplicate samples
were analyzed by A Perkin-2380 atomic absorption spectrophotometry. Samples also were taken
from experimental controls, which contain no biomass (El-Morsy 2004).
Effect of biomass concentration
To evaluate the effect of biomass concentration on the adsorption behaviour of Zn, Cu and
Pb. Biomass concentrations of 100, 200 and 300 mg/l were added to 250 ml Erlenmeyer flasks
separately. Aliquots (50 ml) of heavy metal solution (100, 200 and 300 mg/l) were added to each
flask, and the flasks were left 15 minutes on a rotatory shaker at 180 rpm at room temperature
before being analyzed as above (El-Morsy 2004).
Effect of pH
A Sorption of metal ions by dried mycelial biomass was studied at pH values of 2, 2.5, 3,
3.5, 4, 4.5, 5, 5.5, 6 and 6.5. The samples were shaken at 180 rpm using a rotatory shaker at room
1121
temperature. A fixed biomass of 200 mg/l was added to 50 ml of heavy metal solution containing
Zn, Cu and Pb at an initial concentration of 200 mg/l for 15 minutes. To avoid shifts in pH due to
biomass addition, the pH was adjusted with 0.1N HCl and 0.1N NaOH after the solution had been
in contact with the adsorbent. In the case of Pb, pH was adjusted with 0.1 N HNO3 or 0.1N NH4OH.
Triplicate samples were analyzed as above (El-Morsy 2004).
Time of contact
To determine the optimal incubation time, a fixed adsorbent concentration of 200 mg/l of
fungal biomass was added to 50 ml of heavy metal solution containing an initial metal
concentration of 200 mg/l of Zn, Cu and Pb. Three samples were taken at 5, 10, 15, 25, 35, 60, 120
and 1440 minutes at room temperature (El-Morsy 2004).
Alkali-treatment of the biosorbent
To generate ionic sites without significant modification of the cell wall structure before
sorption, the mycelium was treated with 0.1 M NaOH at 120°C for 6 h and filtered through 0.45
mµ millipore filter paper. The treated mycelium then was washed several times to reach neutral pH
and oven dried at 60°C. After that, Aliquots of 10, 50, 70, 100, 150, 200, 300, 400 and 600 ppm
were added to 250 ml Erlenmeyer flasks with a fixed biomass of the alkali-treated biosorbent (200
mg/l). The flasks then were agitated on a rotatory shaker (180 rpm) for 15 minutes at room
temperature. The pH was adjusted to 4 in the case of Pb and 5 in the case of Zn and Cu (El-Morsy
2004).
Metal biosorption by alkali treated-immobilized fungal biomass
To each well of a percolating plate, 200 mg of alkali–treated biosorbent was added,
followed by a drop of 4% sodium alginate of high viscosity. A drop of 0.25 M CaCl2 was added to
each well separately to form beads. The beads were collected and air dried to yield pellets. Dried
pellets, each containing 200mg biosorbent biomass were added to conical flasks containing aliquots
(50ml) of 10, 50, 70, 100, 150, 200, 300, 400 and 600 mg/l concentrations of zinc sulfate, copper
sulfate and lead acetate. A constant pH of 4 in the case of Pb and 5 in the case of Zn and Cu and a
time of contact of 15 minutes were used for all metal ions at room temperature. After measuring
the residual metal concentration (Cf) in the solution, the beads were collected and regenerated using
diluted acid (0.1 M HCl) followed by 3% bicarbonate. A control experiment was carried out using
mycelium free alginate pellets.
Biosorption of Zn, Cu and Pb from polluted water
10 Samples were collected from canals located near the industrial area of New Damietta in
July 2012. The Samples contained metal ions at different concentrations. Immobilized fungal
biomass (three pellets) was added to 250 ml Erlenmeyer flasks containing 100 ml of polluted water
sample without adjustment of pH. In a second experiment, the test was carried out at pH 4 and pH
5. The flasks then were agitated on a rotatory shaker (180 rpm) for 15 minutes at room temperature.
The residual metal ion concentration was determined by A Perkin-2380 atomic absorption
spectrophotometry.
Biosorption mechanism
To determine the quantity of metal that can be attracted and retained in an immobilized
form, it is customary to express metal uptake (q) by the biosorbent as the amount of metal adsorbed
per unit of biomass. The calculation of the metal uptake (mg/g dry biosorbent) is based on the
material balance of the sorption system. The amount of metal adsorbed by the biosorbent from
solution can be estimated from this formula: q =V (Ci-Cf)/M (Holan and Volesky, 1994) where q is
the metal ion uptake (mg/g), Ci is the initial metal ion concentration (ppm), Cf is the measured final
concentration of the metal ion in the solution (ppm), V is the liquid sample volume (ml) and M is
the starting biosorbent weight (mg).
1122
The sorption–isotherm relationship can be expressed mathematically by plotting q versus Cf.
This first was done in the classical work of Langmuir (1918) who studied activated carbon
adsorption. The linear form of the Langmuir isotherm equation is represented by this equation:
q = Qmax bCf / 1+bCf.
Where Qmax is the maximum amount of metal per gram of biomass corresponding to
saturation of the adsorption sites.The dissociation constant (b) is a coefficient related to the affinity
between metals and biomass.
Results
Occurrence of fungi in the polluted water
Fourty-five fungal species were isolated from polluted water at Damietta’s canals and
drainages located near the industrial area of New Damietta (Table. 1). The majority of species were
mitosporic (37 species of hyphomycetes and three species of agonomycetes). Zygomycetes were
represented by three species while yeast was represented by two species. The frequent species were
Mucor racemosus (50%) followed by Aspergillus flavus (48%), A. niger (36%), A. fumigatus
(30%), Trichoderma Koningii (30%) and Rhizopus oryzae (29%).
Influence of biomass, pH and time of contact on metal uptake
Metal uptake varied with biomass concentration, pH and time of contact. The level of ion
uptake rose with increasing biomass till 200 mg and then decreased with increasing biomass (Fig. 1.
a). Uptake rose with increasing pH up to 4 in the case of Pb and 5 in the case of Zn and Cu (Fig. 1.
b). The maximum uptake of all metals was achieved after 15 minutes and decreased promptly (Fig.
1. c).
Biosorption mechanism
Sorption isotherms of Zn, Cu and Pb by dead biomass of M. racemosus are shown in
(Table. 2). Sorption isotherms represented the distribution of metal ions between aqueous and solid
phases (biomass) when the concentration increases as long as binding sites are not saturated. These
isotherms permit the calculation of the adsorption capacities and dissociation constants of metal
ions. The maximum uptake of Cu (60.13 mg/g), Zn (57.67 mg/g) and Pb (21.97 mg/g) occurred at a
biomass of 200 mg/l.
Metal biosorption by immobilized fungal biomass
Uptake of Zn, Cu and Pb increased after treatment with 0.1 M NaOH at 120oC for 6 h at
biomass of 200 mg/l. Accordingly, NaOH pretreatment was employed in experiments designed to
test the efficiency of alginate-immobilized biomass in enhancing metal uptake. Alginate beads have
the capacity to be laden and remove metal ions from metal solutions and water samples polluted
with heavy metals. The best rate of uptake of alginate beads (q) occurs at 100 ppm for Zn and Pb
and at 150 ppm for Cu. In all heavy metals under study biosorption increased till a definite heavy
metal concentration after which rate of uptake began to decrease by alginate beads. Results revealed
a marked increase in uptake of all tested metals by the alkali treated alginate-immobilized biomass
over free biomass in enhancing metal uptake. The best rate of uptake by alkali treated alginate-
immobilized M. racemosus for Zn occurred at 100 ppm (111.58 mg/g), Cu occurred at 150 ppm
(121.33 mg/g) and Pb occurred at 100 ppm (96.15 mg/g) (Fig. 2).
Biosorption of Zn, Cu and Pb from polluted water
Alkali treated alginate-immobilized biomass showed strong ability for removal of metal ions
from polluted samples in the order Pb > Cu > Zn at pH 4 while at pH 5 Cu > Zn > Pb (Table. 3).
1123
0
10
20
30
40
50
60
70
80
90
50 100 200 300
Biomass(mg)
q(mg/g)
Zn(100 ppm)
Zn(200 ppm)
Zn(300 ppm)
Cu(100 ppm)
Cu(200 ppm)
Cu(300 ppm)
Pb(100 ppm)
Pb(200 ppm)
Pb(300 ppm)
0
20
40
60
80
100
120
2 2.5 3 3.5 4 4.5 5 5.5 6 6.5
pH
rate of uptake(mg/g)
Pb
Cu
Zn
0
50
100
150
510 15 25 35 60 120 1440
contact tim e(m inutes)
rate of uptake (mg/g)
Pb
Cu
Zn
Fig.1 – Effect of biomass (a), pH (b), and contact time (c) by free biomass of M. racemosus. Each
value is the average of three replicates.
Discussion
This study surveyed fungi from polluted water with the goal of identifying new species with
metal-ion biosorbent potential. Fourty–five species were isolated. The most frequent species were
Mucor racemosus, Aspergillus flavus, A. niger, A. fumigatus, Trichoderma koningii and Rhizopus
oryzae. Mucor racemosus is the most frequent fungus in water samples which are polluted with
heavy metals. This suggests that M. racemosus may be heavy metal-resistant fungus as a subject for
the studies reported here.
(a)
(b)
(c)
1124
Table1 Fungi isolated from a polluted water of Damietta's canals and drainages located near the
industrial area of New Damietta.
Species
% of occurrence
Hyphomycetes :-
Aspergillus sulphureus
( Fresenius ) Thom and Church
16
A. candidus
Link
18
A. flavus
Link
48
A. fumigatus
Fresenius
30
A. niger
Van Tiegh
36
A. terreus
Thom and Church
13
A. clavatus
Desmazieres
13
A. cervinus
Massee emend - Neill
18
A. carneus
(Van Tiegh ) Bloohwitz
15
A. terricola Var. Americana
(Marchal)
12
A. wentii
(Wehmer)
10
A. flavipes
(Brain.Et Sart.) Thom et Church
14
Alternaria citri
Elliss et pieree apud.
15
Acremonium guillemattii
(W.Gams)
12
Ac. charticola
(Lindau) Gams
13
Ac. falciform
(Carrion) Gams
15
Ac. strictum
Gams
7
Ampulliferina fagi
M.B.Ellis Spec.nov
9
Botryotrichum piluliferum
Sacc.et March
8
Curvularia pallescens
Boedijin
9
Fusarium moniliforme
Sheldon
11
F. oxysporium
Sehlecht
14
Phialophora richardisae
Nannf
9
Phialophora. cyclaminis
Beyma
7
Penicillium corylophilum
Dierckx
17
P. chermesinum
Biourge
17
p. piscarium
Westling
16
p. camembetri
Thom
15
p. purpurogenum
Stoll
16
p. citrinum
Thom
17
p. notatum
Westling
15
p. simplicissimum
(Oud.)Thom
16
p. candidum
Link
17
p. brevi compactum
Dierckx
19
p. janthinellum
Biourge
18
Trichoderma koningii
Oud
30
Trichoderma piluliferum
Webster and Rifai
27
Agonomycetes:-
Demateaceous sterile mycelium
brown
11
D. sterile mycelium
black
12
Hyaline sterile mycelium
11
Zygomycota:-
Mucor racemosus
Fresenius
50
Rhizopus oryzae
Went et Prinsen
29
Syncephalastrum racemosus
Scholer
12
Yeast:-
Geotrichum candidum .
Link:Fr
12
Trichosporon cutaneum
(De.Beurmann) Ota.
14
The isolated species were classified as very frequent (>20%), frequent (10-20%), or infrequent (<10%) as adapted from
(Tan and Leong 1989).
1125
0
50
100
150
200
250
10 50 70 100 150 200 300 400 600
metal ion concentration (ppm )
rate of uptake (mg/g)
immobilized
alkali treated
biosorbent
alkali treated
biosorbent
ca alginate
0
50
100
150
200
250
300
10 50 70 100 150 200 300 400 600
metal ion cncentration (ppm)
rate of uptake (mg/g)
immobilized
alkali treated
biosorbent
alkali treated
biosorbent
ca alginate
0
20
40
60
80
100
120
140
160
180
200
10 50 70 100 150 200 300 400 600
metal ion concentration(ppm )
rate of uptake(mg/g)
immobilized
alkali treated
biosorbent
alkali treated
biosorbent
ca alginate
Fig. 2 – Biosorption capability of ca alginate, alkali treated and alkali treated alginate-immobilized
Mucor racemosus for (a) Zn, (b) Cu and (c) Pb.
(a
)a
(
b
)
c))
1126
Table 2 Uptake Capacities of various heavy metals by Mucor racemosus derived from the
Langmuir equation (q = Qmax bCf/1+bCf.)
Parameters/metal
Zn
Cu
Pb
A.W.(g)
65.39
63
207.19
Q (mg/g)
57.67
60.13
21.97
Q(mmole/g)
0.88
0.95
0.11
b(mM)
0.3
0.46
2.05
Affinity (1/b)
3.3
2.17
0.49
Q= maximum absorption capacity, b= dissociation constant, A.W= Atomic Weight
Biosorption by free biosorbent and effect of biomass concentration
Fourest & Roux 1992 reported that metal-ion uptake per gram of biosorbent increases as
long as the biosorbent is not saturated. However, uptake values also depend on the nature and the
origin of the biosorbent itself (Luef et al. 1991). In the present study, uptake of Zn, Cu and Pb by
free biosorbent in solution varied depending on the initial metal concentration, biomass
concentration, time of contact and pH. With M. racemosus, the optimal time for biosorption was 15
minutes after contact. This result is similar to that obtained by Volesky & Philips 1995
(Saccharomyces cerevisiae), El-Morsy 2004 (Cunninghamella echinulata), who reported that most
metal biosorption was achieved in 5–15 minutes, followed by residual and slower additional metal
deposition (Tsezos & Volesky 1981), conceivably indicating a secondary metal binding
mechanism. Alike, the maximum uptake by M. racemosus occurred at pH 4 for Pb and at pH 5 for
Cu and Zn with uptake falling with rising pH. The effects of pH on the biosrbent capabilities of
fungal biomass appeared to vary with assay conditions, the particular metal ion and fungal species.
Analogous to results reported here, pH between 4 and 5 was reported as optimal for biosorption of
Zn and Cu by Saccharomyces cerevisiae (Tsezos & Volesky 1981, Tobin et al. 1984, Volesky &
Philips 1995) found that pH near 4 was optimal for metal uptake by Rhizopus arrhizus. In contrast,
Luef et al. 1991 reported that biosorption of Zn by mycelium of A. niger, Penicillium chrysogenum
and Clavicipis paspali rose with increasing pH up to 9. Fourest and Roux 1992 reported that the
optimal uptake of Zn and Pb by Rhizopus arrhizus was achieved at a neutral pH and pH 5,
respectively. Lewis & Kiff 1988 found that acidic pH reduced metal biosorption by the latter, a
reduction that could be attributed to the precipitation of metal ions.
Biosorption mechanism
The sorption process involves biomass as a solid phase and a liquid phase containing metal
ions, with ion distribution between solid and liquid phases determined by the affinity of biomass for
metals. The quality of the biosorbents material is evaluated in terms of how much metal it can
attract and retain in an immobilized form. It is customary to determine metal uptake (q) by the
biosorbents as the amount of metals bound by the unit of biomass. Sorption isotherm followed the
typical Langmuir adsorption pattern (Ruthven 1984). The results presented here for M. racemosus
are consistent with the Langmuir – isotherm model (Fourest & Roux 1992).
Alkali treatment of biosorbents pretreatment of the biomass was performed following the
work of (Kapoor & Viraraghavan 1997, 1998 a & b, Kapoor et al. 1999). Alkali treatment
improved the capacity of the M. racemosus biosorbents to chelate metal ions, especially the higher
biomass of 200 mg/l. The higher affinity might be attributed to the chitin and chitosan content of
the fungus cell wall, exposed after NaOH treatment. NaOH appears to remove amorphous
polysaccharides from the cell wall, generating accessible space within the β-glucan–chitin skeleton
and hence permitting metal ions to precipitate on this surface (Tsezos & Volesky 1981, Fourest &
Roux 1992). Similaraly formaline pretreatment significantly improved the surface active site that
actually participitates in Cu biosorption (Kapoor & Viraraghavan 1997, 1998 a & b, Kapoor et al.
1999).
1127
Table 3 Biosorption efficiency (%) of Zn, Cu and Pb from polluted water by alginate pellets of
alkali treated-immobilized Mucor racemosus.
Pb
Cu
Zn
Parameters/metal
96.15
81.11
80.55
pH=4
83.25
95.88
95.04
pH=5
39.37
63.10
56.65
Control (without pH
adjustment)
Kapoor et al. 1999 reported that both Langmuir and Freundlich isotherm data at pH 5 at Cu
biosorption by NaOH pretreated Aspergillus niger biomass were statistically significant at 95%
confidence level. The alkaline treatments including sodium hydroxide, potassium hydroxide,
alkaline detergents or other alkaline reagents ruptures the cell walls of microbes and exposes
additional functional groups for metal ion binding. The residual alkalinity may result in the
hydrolysis of certain metals, thus enhancing the biosorption capacity of the biomass (Brierley
1990). The biomass of Aspergillus niger pretreated with Na2CO3 (0.2 N) exhibited the maximum
biosorption capacity (20.82 mg/g) for Cu.
Treatment of A. niger with NaOH (0.2 N and 0.5 N ) yield equivalent adsorption capacity
(18.81 mg/g) the biosorption capacity of (16.5 mg/g ) recorded for fungal biomass pretreated with
NaHCO3 (0.2 N ) and detergent was observed to reduce up to 22% in comparison to maximum
recorded potential of >37% in alkali pretreatment (Javaid et al. 2011).
Baik et al. 2002 reported that Aspergillus sp. has the ability to absorb maximum level of Cu
when treating the cell fraction with NaOH. Volesky & Philips 1995 explained this is due to
microbial biomass consisting of poor mechanical strength and little rigidity. However, biosorbents
are hard enough to withstand and the application of pressures, water retention capacity, porous and
transport to metal ion sorbate species.
Physical and chemical treatments used to enhance the metal uptake capacity of the biomass,
which led to removal, hiding or exposing chemical groups that binding or exchange with the
adsorbed metal ions (Saleh et al. 2009). Similar enhancement in metal uptake capacity of the fungal
biomass regarding alkali pretreatment was recorded by (Yan &Viraraghavan 2000, El-Morsy 2004,
Das et al. 2007). It could be due to chemical modifications of the cell wall components. The
modification of biomass probably destroys autolytic enzymes that cause purification of biomass and
remove lipids and proteins that mask the reactive sites (Muraleedharan &Venkobachar 1990).
Immobilization of the biosorbents
Immobilization has been reported to enhance the capacity of fungal biomass for chelating
metal ions (Lewis & Kiff 1988, Yousef 1997). Here, alginate immobilization of alkali treated
biosorbents beads resulted in a nearly two fold increase in metal-ion uptake over free biosorbents.
Alginate carboxyl groups are known to play an important role in metal binding (Kuyucak &
Volesky 1989). Previous studies have revealed a high capacity of alginate-biomass beads to remove
metal ions from polluted water (El-Morsy 2004).
In this study, up to 96.15%, 81.11 % and 80.55% of Pb, Cu and Zn were removed at pH 4
respectively. While at pH 5 up to 95.88 %, 95.04 % and 83.24% of Cu, Zn and Pb were removed
respectively. Kapoor & Viraraghavan 1998b used A. niger biomass immobilized in polysulfone
polymer in the form of spherical beads to remove Zn, Cu and Pb ions from industrial waste water.
A packed bead column containing A. niger beads removed 58 % Pb, 38 % Cu and 16 % Zn from the
culture in 12 h. Biomass immobilized on polyacrylamide similarly was non immobilized biomass
for metal biosorption (Yousef 1997). The uptake of the heavy metals (Cu, Co and Fe) was
examined in the immobilized column experiments (Rhizopus delemar immobilized on polyurethane
foam). More than 92% heavy metal removal from a mixed solution was achieved during the 5
cycles (Kolishka Tsekova & Galin Petrov 2002). More recently, biosorption of metals by
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immobilized cell system has been used efficiently for removal of metals from industrial effluent
(Gadd &White 1993, Gulay et al. 2003, Yakup et al. 2004, Sun et al. 2010, Hemambika et al. 2011,
Tan & Ting. 2012, Ahemad & Kibret 2013). Cells with such abilities are immobilized either as
entrapped biomass or as a biofilm to form a system for treating waste water known as a bioreactor
(Qureslii et al. 2001). Immobilized cells of Kluyveromyces marxianus allowed for high removal
capacity for Cu, Co and Zn where 1.6, 1.5 and 1.3 fold increase were recorded compared to free
cells (Yousef 1997).
The biosorption of immobilized cells of Aspergillus sp. was 60.94% of Cu, Penicillium sp.
was 97.21% of Cd and Cephalosporium sp. was 73.27% of Pb; whereas the dead cells of
Aspergillus sp. was 46.91% of Cu, Penicillium sp. was 95.27% of Cd and Cephalosporium sp.
was 70.67% of Pb. All these results reveal that the adsorption capacities of the immobilized fungal
cells were greater than that of dead cells (Hemambika et al. 2011). Johncy Rani et al. 2010 found
similar adsorption capacity of Cu, Cd and Pb in the immobilized cells of Bacillus sp., pseudomonas
sp. and Micrococcus sp. respectively. Leusch et al. 1995 explained this is because dead fungal cells
consist of small particles with low density, poor mechanical strength and little rigidity. Hence, the
immobilization of biomass is necessary on before subjecting to biosorption. Holan & Volesky 1994
explained that immobilized cells offers many advantages including better reusability, high biomass
loading and minimum clogging in continuous flow systems. Adsorption of heavy metals was also
dependent on cell density in calcium alginate beads. The results obtained for the uptake of heavy
metals under study show us that the metal accumulation by Mucor racemosus is a chemical,
equilibrated and saturable mechanism. Thus, adsorption increases when the initial metal
concentration rises as long as the binding sites are not saturated.
Zygomycetes sp such as Mucor sp, Rhizopus arrhizus and Absidia orchidis generally are
reported to be efficient biosorbent agents (Lewis & Kiff 1988, Fourest & Roux 1992, Mueler et al.
1992, Holan & Volesky 1995). The work reported here demonstrates M. racemosus to be as
effective as Cunninghamella echinulata and Rhizopus sp. This based on the finding that
immobilized biomass of C. echinulata was able to remove ions from a natural environment up to 95
% (El-Morsy 2004). It was also found that maximum copper removal by Rhizopus arrhizus of 98%
was obtained using the biosorption kinetics of copper under optimum conditions. (Preetha &
Viruthagiri 2007).
Conclusion
Biomass of M. racemosus efficiently is able to remove Zn, Cu and Pb from a solution. This
capacity was enhanced when the biomass was alkali treated and immobilized. The fungus in its
immobilized form also was able to remove ions from a natural environment up to 96 %, therefore
the fungus is proposed as an effective biosorbent for removal of heavy metals in waste water
treatment.
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