Bioresource Technology 218 (2016) 388396

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Multiple heavy metal removal using an entomopathogenic fungi

Beauveria bassiana
Deepak Gola a, Priyadarshini Dey a, Arghya Bhattacharya a, Abhishek Mishra a, Anushree Malik a,,
Maneesh Namburath a, Shaikh Ziauddin Ahammad b
a
Applied Microbiology Lab, Centre for Rural Development and Technology, Indian Institute of Technology, Hauz Khas, Delhi, India
b
Department of Biochemical Engineering & Biotechnology, Indian Institute of Technology, Hauz Khas, Delhi, India

h i g h l i g h t s g r a p h i c a l a b s t r a c t

High multimetal (84%) removal

compared to individual metal (61
75%) by B. bassiana.

Relative metal affinity changed under
multimetal as compared to single
metal.

AFM revealed changes in surface
roughness due to metal toxicity.

Potential strain for multimetal
removal from contaminated
wastewater.

a r t i c l e i n f o a b s t r a c t

Article history: Towards the development of a potential remediation technology for multiple heavy metals [Zn(II), Cu(II),
Received 19 May 2016 Cd(II), Cr(VI) and Ni(II)] from contaminated water, present study examined the growth kinetics and heavy
Received in revised form 21 June 2016 metal removal ability of Beauveria bassiana in individual and multi metals. The specific growth rate of B.
Accepted 24 June 2016
bassiana varied from 0.025 h1 to 0.039 h1 in presence of individual/multi heavy metals. FTIR analysis
Available online 27 June 2016
indicated the involvement of different surface functional groups in biosorption of different metals, while
cellular changes in fungus was reflected by various microscopic (SEM, AFM and TEM) analysis. TEM
Keywords:
studies proved removal of heavy metals via sorption and accumulation processes, whereas AFM studies
Entomopathogenic fungi
Multi metal
revealed increase in cell surface roughness in fungal cells exposed to heavy metals. Present study delivers
FTIR first report on the mechanism of bioremediation of heavy metals when present individually as well as
SEM-EDX multi metal mixture by entomopathogenic fungi.
TEM 2016 Elsevier Ltd. All rights reserved.

1. Introduction
Increased industrialisation activities have contributed largely
towards the introduction of heavy metals and other pollutants into
the environment (Bhattacharya et al., 2015; Gupta et al., 2014;
Mishra et al., 2014; Shah, 2014). Industries directly discharge
heavy metal containing waste into the river and drains which
Corresponding author
E-mail addresses: anushreemalik@yahoo.com, anushree@rdat.iitd.ac.in (A. Malik).
enters the food chain via vegetables irrigated with contaminated
water (Nguyen et al., 2013). As a result, heavy metals accumulate
in human body thereby causing potential health hazards. Exposure
to these toxic metals is associated with many serious diseases,
including autoimmune disorder, digestive disorder, heart disor-
ders, liver, kidney, stomach and lung cancer (Yadav et al., 2010).
Hence, heavy metals must be removed or transformed to less toxic
forms in wastewater before being discharged to the environment
or before using it for irrigation. Physical and chemical processes
are conventionally used for removing heavy metal, but these

http://dx.doi.org/10.1016/j.biortech.2016.06.096
0960-8524/ 2016 Elsevier Ltd. All rights reserved.
 D. Gola et al. / Bioresource Technology 218 (2016) 388396
processes are not economical when the concentration of heavy
metal is very low (Li et al., 2013). Biological processes are suitable
for remediating single or binary metal solutions (Bulgariu and
Bulgariu, 2012; Khodabakhsh et al., 2011; Mishra and Malik,
2012). The efficiency of these processes decreases when employed
for multiple heavy metal removal.
Majority of the studies have targeted bioremediation
technology for removal of a single metal or at the most binary
metal mixture because microbes are unable to efficiently deal with
multiple heavy metals at the same time. This is due to the fact that
the individual metals in mix can interact in synergistic, antagonis-
tic and non-interactive manner to influence the resultant toxicity.
In most of the studies, synergistic effect was observed in multiple
metal ion mixture, whereas few studies discussed about the antag-
onistic effect (Gola et al., 2016). Moreover, in multi metal mixture
some metals with high affinity for the biomass are effectively
removed but others may still be retained in the solution. For exam-
ple, Bacillus sp. L14 could uptake up to 75.7% and 80.4% of Cd (II)
and Pb (II) but only 21.2% of Cu (II) was removed from initial con-
centration of 10 mg L1 (Guo et al., 2010). Aspergillus niger could
also remove significant quantities of Cu(II) and Pb(II) from growth
media, but was less resistant against Cr(VI) (Dursun et al., 2003).
Mishra and Malik (2012) observed 78100% metal removal for Cr
(III), Cu(II) and Pb(II), but only 42% of Ni(II) could be removed from
synthetic solution, by Aspergillus lentulus. On the other hand,
8994% metal removal was attained for Ni(II), Cu(II), Cr(VI) and
Zn(II) by Saccharomyces cerevisiae at pH 6 (Machado et al., 2010).
Beauveria bassiana is an anamorphic entomopathogenic species
of fungi which is a natural enemy of insects and arachnids (Khalid
et al., 2011). Large volume of literature is available on its insectici-
dal properties but relatively few reports exist on metal remedia-
tion using entomopathogenic fungi. Kameo et al. (2000) studied
the production of metallothioneins (cysteine rich metal binding
protein) in the presence of Cu(II) and Cd(II) using a Cu(II) resistance
Beauveria bassiana strain. Upon heavy metal exposure, various fun-
gal strains synthesize metallothioneins to provide resistance
against heavy metal toxicity. Khalid et al. (2011) studied the
bio-sorption of Pb(II) and Cd(II) (under single metal exposure)
using the dead biomass of B. bassiana and observed metal uptake
capacity up to 46 mg g1 and 86.3 mg g1 for Pb(II) and Cd(II),
respectively. In spite of such lab scale studies, very less is known
about the heavy metal bioremediation role of fungi in actual envi-
ronmental conditions. Metal tolerant fungi, Beauveria bassiana and
Rhodotorula mucilaginosa, isolated from constructed wetlands were
found to possess high MIC for Zn(II) and Pb(II) (Scholes et al.,
1999). Purchase et al. (2009) demonstrated that these strains
could accomplish Zn(II) and Pb(II) accumulation at extremely
low temperature (4 C) as well thereby establishing their
importance for round the year metal removal in wetlands. How-
ever, in none of the studies, simultaneous uptake of multiple
metals by Beauveria species or other entomopathogenic fungi is
described.
Recently, AFM (Atomic Force Microscopy) has been used to
detect nano-mechanical properties induced to the
micro-organism, mammalian cells and nanoparticles in different
conditions. However, limited studies have been conducted to
determine the changes in the nano-mechanical properties of fungal
cell walls (Das et al., 2008). In view of above, the objective of the
present study was to investigate the ability of environmentally
benign fungal strain to deal with the individual metal as well as
multiple metals stress and to understand the impact of metal
(single versus multiple) exposure on morphological characteristics
and surface properties of the fungal strain. Briefly, a fungal isolate
with great potential for simultaneous uptake of multiple metals
has been described.
389
2. Materials and methods
2.1. Microorganisms, growth conditions and minimum inhibitory
concentration (MIC)
Beauveria bassiana was previously obtained by from Institute of
Microbial Technology (Chandigarh, India). Tolerance to heavy met-
als [Cu(II), Cr(VI), Cd(II), Zn(II) and Ni(II)] was determined in terms
of the MIC of metals. The strain was grown in composite media
(NH4NO3, 0.5 g L1; MgSO47H2O, 0.1 g L1; K2HPO4, 0.5 g L1;
NaCl, 1 g L1; Glucose, 10 g L1; Yeast extract, 2.5 g L1) having dif-
ferent concentration of heavy metal ranging from 25 to 300 mg L1
and pH was adjusted to 6.57.0. Metal containing media was
inoculated with fungal spore (106 spores mL1) and was incubated
at 30 C at 150 rpm. Growth was monitored after every 24 h for
5 days.
2.2. Metal solutions preparation
Metal stock of 1000 mg L1 was prepared by dissolving their
respective salts CuSO4H2O, ZnSO47H2O, K2Cr2O7, Ni(NO3)2 and
CdSO48H2O in double distilled water, then diluted to the desired
concentration prior to use in the experiments.
2.3. Effect of metal on growth kinetics and metal removal mechanism
The removal of heavy metal via growing cell is called as
bioaccumulation. The influence of heavy metal on the growth
and bioaccumulation property of fungus was investigated in pres-
ence of 30 mg L1 individual heavy metals as well as in multi metal
mixture [6 mg L1 Cu(II), Cr(VI), Cd(II), Zn(II) and Ni(II) each]. 1 mL
Beauveria bassiana spore suspension (106 spores mL1) was inocu-
lated in 100 mL composite media containing 30 mg L1 total metal
concentration. Flasks were removed at regular intervals and
samples were analysed for residual metal concentration, glucose
concentration and biomass produced. Biomass was filtered
through Whatman No.1 paper. Further, the filter paper was dried
at 60 C to a constant weight and gravimetric method was used
to determine the fungal biomass. The specific growth rate (l) of
fungus was calculated as:
1 dX
l
X dt
where, X is biomass concentrations (g L1) at time t (h). The slope of
the plot of ln X vs t gave the value of l.
To estimate biosorption capacity of fungal biomass in multi
metal mixture, nitric acid (0.05 M) was used to desorb the metal
biosorbed on the surface. Metal laden biomass was obtained by
filtration and was contacted with 100 mL nitric acid. The mixture
was agitated at 150 rpm for 30 min. Subsequently, supernatant
was obtained and analysed for different heavy metals. To calculate
the bioaccumulated metal concentration, the amount of biosorbed
metal was subtracted from the total metal removed.
2.4. Effect of initial heavy metal concentration
The effect of different concentrations (30100 mg L1) of indi-
vidual heavy metals [Cu(II), Cr(VI), Cd(II), Zn(II) and Ni(II)] and
metal mix on growth and metal removal efficiency of Beauveria
bassiana was evaluated by inoculating the flask with 1% spore sus-
pension containing composite media and individual heavy metal.
390 D. Gola et al. / Bioresource Technology 218 (2016) 388396
2.5. Effect of pH and temperature on metal removal in multi metal
mixture
To study the influence of pH on heavy metals removal at
30 mg L1 (initial concentration), the initial pH of the composite
media was adjusted to the desired value ranging from 4 to 9, by
using sterile H2SO4 and NaOH solutions. All the flasks were inocu-
lated with a spore suspension (106 spores mL1) of the fungus and
incubated at 30 C and 150 rpm for 5 days. To determine the influ-
ence of temperature on multi metal removal, inoculated flasks
were incubated at three different temperature (20 C, 30 C and
40 C) with 150 rpm for 5 days. Positive controls were run at each
pH and temperature where flasks containing composite media
without metal addition were inoculated and incubated under the
conditions described above.
2.6. Measurement of residual metal concentration
Ten mL of the samples from flask were taken aseptically and
centrifuged at 10,000 rpm for 10 min. The supernatant liquid was
digested in microwave digester (160 4 C within 10 min and
165170 C for 10 min for reaction) and analysed for metal ions
via Microwave plasma-atomic emission spectrometry (Agilent
4200 MP-AES).
2.7. Estimation of glucose content
Glucose was analysed in the samples using DNS (using di-nitro
salicyclic acid) reagent. DNS was prepared by dissolving 3,5
di-nitro salicyclic acid (1 g) and NaOH (100 mL of 1%), Phenol
(200 mg), sodium sulphite (50 mg) and rochelle salt solution
(40%) gradually in distilled water. The resulting solution was kept
in dark to avoid degradation due to light. Supernatant was
obtained by centrifugation. To 1 mL of sample, 1 mL of distilled
water and 3 mL DNS reagent were added. The solution was kept
for boiling at 90 C for 10 min in water bath. After cooling 10 mL
distilled water was added. The resultant solution was read at
540 nm wavelength using a spectrophotometer (Kaushik et al.,
2010).
2.8. Morphological analysis
2.8.1. FTIR (Fourier transform infrared spectroscopy)
The functional group responsible for metal uptake on Beauveria
bassiana were analysed by FTIR. Biomass from the flask containing
30 mg L1 heavy metal (individual metal or metal mixture) as well
as control were obtained and lyophilised. Next, 1 mg fine lyophi-
lised fungal biomass was mixed uniformly with 100 mg KBr. FTIR
spectra was obtained in the range of 6504000 cm1 with 3351
scans (Perkin Elmer One spectrum-spectrum v5.0.01).
2.8.2. AFM (atomic force microscopy)
For AFM imaging, fungal pellet was harvested from the growth
media and washed with deionised water. After washing, pellet was
suspended in 50 mM citrate-phosphate buffer (pH 3) for condition-
ing. Pellet from the citrate buffer was obtained by centrifugation
and placed on the clean glass cover slip for 30 min, followed by
repeated washing with deionised water and then air dried. AFM
micrographs were recorded with silicon cantilever with force
constant 0.220.77 N/m, tip height 1012 nm in the contact mode
via vecco metrology nanoscope IIIa.
The root-mean-square average of the surface roughness (Rrms)
was measured with the following expression:
q
X Zn(II)
Rrms Zi  Zav g2=Np
where Zi is the current Z value, Zavg is the average of the Z values
within the given area, and Np is the number of point within the
given area (Das et al., 2008).
2.8.3. SEM-EDX (scanning electron microscopy-energy dispersive
X-ray spectroscopy)
In order to observe how binding of the metal ions on the cell
surface alter its cell-surface morphology, SEM was performed via
ZEISS EVO 50. Further, to confirm the presence of metal ions on
the cell surface of the fungus, EDX was employed with the same
instrument. In order to perform the analysis, the fungus pellet from
each flask were fixed with 2.5% glutaraldehyde in phosphate buffer
for 30 min and washed three times in 0.1 M phosphate buffer
solution (pH 7.2). Now each suspension was serially dehydrated
with 25%, 50%, 75%, 90% and 100% ethanol, respectively. For SEM,
samples were gold-coated by cathodic spraying and observed
under a scanning electron microscope. The SEM observation was
done under the following analytical condition: EHT = 20.0 kV,
Signal A = SE1. For EDX, samples were carbon coated and observed
under the same scanning electron microscope.
2.8.4. TEM (transmission electron microscopy)
For TEM, fungal pellet was obtained by centrifugation at 1000g
for 10 min. Fungal pellet was washed with phosphate buffer saline
(pH 6.8) and subsequently fixed in 1% glutaraldehyde & 2%
paraformaldehyde for 1218 h at 4 C. After fixation, pellets were
washed again using PBS buffer. Post washing fixation was done
using osmium tetra oxide (1%) for 2 h at 4 C. Pellet was than dehy-
drated using acetone and embedded in epon-araldite. Ultrathin
sections of 6080 nm thickness were cut using an ultramicrotome
(Leica EM UC 6). The sections were stained in alcoholic uranyl
acetate and lead citrate for 10 min, before examining the grids in
a transmission electron microscopy (JEOL JEM-2100F) operated at
6080 kV.
3. Results and discussion
3.1. Minimum inhibitory concentration (MIC)
Minimum inhibitory concentration of different heavy metals
[Cu(II), Cr(VI), Cd(II), Zn(II) and Ni(II)] for B. bassiana ranged
between 100 and 250 mg L1 with highest MIC recorded for Zn
(II) followed by Cd(II) as summarized in the Table 1. Although
the strain did not display remarkably high MIC against test metals
as depicted by other recently reported (Mishra and Malik, 2014)
fungi like Aspergillus lentulus [>12,000, 5000, 850, 550 and
300 mg L1 for Cr(III), Pb(II), Cu(II), Cr(VI) and Ni(II), respectively]
or Aspergillus terreus [12,000 and 600 mg L1 for Cr(VI) and Cu(II),
respectively], the results still retain significance due to its ability
to deal with milder concentration of multiple metals. Our recent
study showed that concentration of above heavy metals in Yamuna
river varied from 0 to 2.22 mg L1, with Zn(II) being recorded at
highest concentration (Bhattacharya et al., 2015). Hence, it can
be inferred that this B. bassiana strain will survive under the
moderately polluted conditions encountered in the environment.
Table 1
MIC of heavy metal for Beauveria bassiana.
Metal MIC(mg L1)
Cu(II) 100
Ni(II) 100
Cd(II) 200
250
Cr(VI) 100
 D. Gola et al. / Bioresource Technology 218 (2016) 388396 391

3.2. Effect of metal on growth kinetics and metal removal mechanism
Fig. 1 shows the pH profile, rate of glucose utilization and bio-
mass produced by B. bassiana in the absence and presence of individ-
ual heavy metal as well as metal mix (30 mg L1 concentration) in
the growth medium. Decrease in the pH (6.54.42) was observed
during the lag phase in the growth medium with or without heavy
metal. It was observed that lag phase was very short and limited
to 6 h in the absence of heavy metal as compared to 1224 h in the
presence of heavy metal. The increment in the lag phase indicates
the adaptation of the spore with the environment, contributing to
the slow rate of glucose consumption in presence of heavy metal
as shown in the Fig. 1. Similar increase in lag phase in presence of
heavy metals was observed with Trichoderma viride (Anand et al.,
2006) and A. lentulus as well as A. terreus (Mishra and Malik, 2012).
At the end of 120 h, maximum biomass was produced in absence
of any metal (5.6 g L1) which decreased to some extent in presence
of Zn(II) (5.3 g L1), Ni(II) (5.0 g L1), Cu(II) (4.9 g L1), Cr(VI)
(4.8 g L1) and multi metal mixture (4.8 g L1). A more severe reduc-
tion in biomass production occurred in presence of Cd(II) (4.2 g L1)
alone. Table 2 shows that the specific growth rate in the absence of
heavy metal was 0.118 h1 which was reduced to 0.0250.037 h1
in presence of heavy metals. Hence, all the tested metals induced
growth inhibition to varying extent. Lpez Errasqun and Vzquez
(2003) also observed that under single metal exposure, Cd(II) caused
highest reduction in biomass growth of Trichoderma atroviride as
compared to Cu(II) and Zn(II). However, substantial decline in
biomass occurred when T. atroviride was cultured with zinc plus

Fig. 1. Growth kinetic and removal of heavy metal by Beauveria bassiana with (A) in absence of metal; (B) at 30 mg L1 of Cd (C) at 30 mg L1 of Cr (D) at 30 mg L1 of Cu; (E)
at 30 mg L1 of Ni; (F) at 30 mg L1 of Zn and (G) at 30 mg L1 of Multi Metal Mix. [individual heavy metal concentration removed (G1)].
392 D. Gola et al. / Bioresource Technology 218 (2016) 388396
Table 2
The effect of heavy metal on the specific growth rate and percentage of metal removal
by Beauveria bassiana at 30 mg L1 initial heavy metal concentration.
Metal Specific growth rate l (h1) Maximum metal removal (%)
Control 0.118
Cu(II) 0.025 74.13
Ni(II) 0.039 75
Cd(II) 0.036 63.4
Zn(II) 0.032 67.8
Cr(II) 0.037 61.13
Multi metal 0.034 84.53
cadmium or with a combination of the three metals. A synergistic
interaction between zinc and cadmium was noted resulting in
greater toxicity of the combination. In the present study, no such
synergistic interaction was observed among tested metals.
The removal of Cu(II), Ni(II), Cd(II), Zn(II), Cr(VI) and multi metal
with time is also shown in Fig. 1. Under single metal exposure,
least removal was observed for Cr(VI) (61.1%) followed by Cd(II)
(63.4%) and Zn(II) (67.8%). In spite of the fact that Zn(II) supported
maximum biomass production, corresponding removal could not
be obtained. In fact, the removal of Zn(II) was poorly correlated
with biomass growth (Fig. 1F) with substantial removal (35.9%)
within first 30 h when the biomass growth was still in the lag
phase. Similar behaviour was observed for Cr(VI) removal where
substantial metal removal occurred in lag phase of biomass
growth. This indicates that Zn(II) and Cr(VI) removal mechanism
may not be directly correlated with increase in biomass surface
area or metabolic activity. Significantly higher metal removal
(7475%) was observed for Ni(II) and Cu(II), both of which sup-
ported moderate biomass production (4.95.0 g L1). Highest total
metal removal rate, synchronized with biomass growth was
observed in presence of multi metal. At the end of 120 h, 84.5%
total metal was removed while the biomass obtained (4.8 g L1)
was lesser than that obtained in presence of single metals such
as Cu(II), Ni(II) and Zn(II). This might be due to the decrease in
concentration of individual heavy metal and more availability of
binding site on the surface of fungus. Similar higher removal
capacity in multi metal ion solution [Cr(VI), Ni(II) and Cd(II)] was
observed with Aspergillus sp. and Penicillium sp as compared to
the single metal ion (Ahmad et al., 2006).
The results are contrary to those reported earlier where
reduction in metal uptake capacities was recorded in presence of
combination of metal ions as compared to single metal (Chang
and Huang, 1998). The authors suggested that the selective accu-
mulation of heavy metals by micro-organisms is determined by
interionic competition in which metal cations compete for the
binding sites on the cell wall, thereby reducing the metal uptake.
However, similar to our results, Lpez Errasqun and Vzquez
(2003) observed stimulation of uptake by T. atroviride, when other
cations were present in the medium. In their study, a doubling of
uptake was observed for copper in a three metal combination,
compared to copper alone. Pan et al. (2009) investigated the
removal of heavy metal in multi metal mixture containing Cd(II),
Cu(II), Zn(II) and Pb(II) using growing Penicillium sp. A1 and
Fusarium sp. A19. The authors concluded that in multi metal
solution various type of interaction (such as synergetic/antagonis-
tic/no interaction) occur between metal ion and microorganism.
The complexity of such situations can be further gauged by the
results reported by Aikel and Alp (2009). The authors observed
that the bio accumulation process of Cu(II) and Ni(II) metal ions
was reduced by the presence of increasing concentrations of the
other metal ion in the media, compared with the single-metal
systems. Hence, the individual action of Cu(II) and Ni(II) metal ions
on the bioaccumulation of Rhizopus delemar was reported to be
antagonistic in nature. On the other hand, the total bio accumu-
lated metal ion concentrations as well as the total bio accumulated
metal ion quantities per unit mass of biomass were on higher side
than those obtained in the growth media containing Cu(II) and Ni
(II) metal ions alone at the same concentrations. Hence, the total
combined effects of Cu(II) and Ni(II) metal ions on the bioaccumu-
lation of R. delemar were suggested to be synergistic in nature,
which was attributed to the twofold increment of the total metal
ion concentration in a mixture containing Cu(II) and Ni(II) metal
ions compared with the single metal ion concentration of Cu(II)
or Ni(II) ion alone resulting in a strong driving force. In the present
study, the total metal concentration remained same in single as
well as multiple metal exposure, yet the total uptake was higher
in the latter case.
The above discussion suggests that no specific and universal
trend can be prescribed between biomass growth and metal
removal. This is because the uptake of metals by biomass is also
a function of affinity of a particular metal at a given concentration.
While some metals do not induce severe toxicity and result in
ample biomass production, low affinity to biomass under given
conditions would result in its lower removal. Overall, the trend
of metal removal under single metal exposure was Multi
metal > Ni(II) P Cu(II) > Zn(II) > Cd(II) > Cr(VI). However, the rela-
tive trend of metal removal under multiple metal exposure chan-
ged with Cr(VI) showing highest metal removal (99.9%) followed
by Cd(II) (95.3%) and Cu(II) (92%) while Ni(II) (71.3%) and Zn(II)
(65.3%) showed lower removal. In order to understand the mecha-
nism of multi metal removal, active and passive uptake of heavy
metal were investigated (Fig. 2). It was observed that substantial
fraction of Zn(II) and Cr(VI) removal occurred through biosorption
mechanism. Up to 41.8% removal of Zn(II) and 37.9% removal of Cr
(VI) was due to biosorption. Whereas, uptake of Cd(II) was mostly
dependent on bioaccumulation i.e. 88.1% as compared to the
biosorption (11.8%), followed by Ni(II). The above results are in
agreement with the TEM observations. Hence, it appears that
heavy metal removal is being affected by both biosorption and
bioaccumulation mechanisms simultaneously.
In multi metal ion system, elemental properties of heavy metals
such as atomic weight, atomic or ionic radius and electronegativity
affect the overall removal efficiency. Pakshirajan and
Swaminathan, (2009) observed removal of Pb(II) was on higher
side as compared to Cu(II) and Cd(II), due to higher ionic radii of
Pb(II) as compared to other heavy metals. Similarly, in present
study, the heavy metal removal follows the trend based on ionic
radius i.e. Cd(80 pm) > Cu(73 pm) > Ni(70 pm) > Zn(60 pm) except
for Cr(VI) (58 pm) that showed maximum removal. Similar trend
of maximum removal of Cr(VI) in the presence of other metal ion
such as Cu(II) and Cd(II) was observed with Rhizopus arrhizus
(Sag et al., 2002). The exact reason for the observed change in
Fig. 2. Passive and active heavy Metal uptake by B. bassiana in multimetal mixture.
 D. Gola et al. / Bioresource Technology 218 (2016) 388396 393

relative affinity of metals under single metal and multi metal specific metal ion uptake i.e. 1 mg g1, and 0.083 mg g1 for Cu
exposure needs to be investigated further. (II) and Ni(II), respectively, for fungal isolates at different initial
The above discussed studies suggest that no clear cut evidence concentration ranging from 10 mg L1 to 50 mg L1.
is available depicting the impact of multi metals versus individual Different metals depicts different trends in the present study.
metals. Some studies indicates variable impacts while others Metal uptake increases significantly with the increase in metal con-
suggest synergetic/antagonistic interaction among metal to centration for Cu(II) and Zn(II). While, in case of Cr(VI), it marginally
increase/decrease toxicity. In view of this, the ability, of B. bassiana increased in spite of reduction in biomass. In multi-metal mix, a
strain to provide ample biomass and show highest removal for tremendous increase in specific meal ion uptake was observed from
multi metals is significant. Therefore, it is worthwhile to 5.2 mg g1 at 30 mg L1 to 19.2 mg g1 at 100 mg L1. Ni(II) and Cd
investigate the effect of increasing metal concentration to ensure (II) displayed interesting behaviour. For Ni(II), specific heavy metal
satisfactory performance of the strain in varying metal stress uptake increases initially from 4.4 mg g1 at 30 mg L1 to
encountered at different environmental sites. 5.8 mg g1 at 50 mg L1 and then decreases to 4.5 mg g1 for
100 mg L1. In spite of minimal reduction in biomass, the Ni(II)
uptake declined with the increase in metal concentration. This indi-
3.3. Effect of initial metal ion concentration
cates that although Ni(II) is less toxic, the affinity of biomass for Ni
(II) is low. The possible reason for this could be un-optimal media
Table 3 shows metal uptake capacities of Beauveria bassiana at
pH, which range from 5 to 7. Dnmez and Aksu (2001) observed
different initial heavy metal concentration in the growth medium.
that removal of Ni(II) is pH dependent and optimal pH for Ni
As initial concentration of heavy metal was increased from
removal by Candida sp. is 4; below and above this pH the removal
30 mg L1 to 100 mg L1, maximum reduction in biomass occurred
of Ni(II) declined considerably. In case of Cd(II) also, metal uptake
in presence of Cd(II) (61.9%) followed by multi metal (56.2%) and Cr
declined almost 41.6% along with significant decline in biomass
(VI) (50.2%), indicating Cd(II) to be more toxic in comparison with
from 4.2 g L1 (at 30 mg L1) to 1.61 g L1 (at 100 mg L1).
other heavy metals. Lpez Errasqun and Vzquez (2003) also
Above results show that B. bassiana can be successfully used for
observed that toxicity of Cd(II) for T. atroviride was on higher side
heavy metal remediation from wastewater contaminated with
as compared to Cu(II) and Zn(II). The present investigation has
multiple heavy metals. The strain can be applied for treatment of
shown 19.5% reduction in biomass with Ni(II) at 100 mg L1. The
industrial (tannery/textile dyeing) effluents, where selected metals
specific metal ion uptake increases with the increase in the initial
are released. Also, it is highly appropriate for treatment of
heavy metal ion concentration except for Ni. The maximum speci-
irrigational waters (from river bodies/drains) which contains low
fic metal ion uptake was determined as 12.2 mg g1 for Cu(II),
concentration of multiple metals. Since the metal removal capacity
13.5 mg g1 for Cd(II), 12.2 mg g1 for Zn(II) and 11.3 mg g1 for
is considerably influenced by pH and temperature (Purchase et al.,
Cr(VI) at 100 mg L1 initial heavy metal concentration. In the multi
2009), the effect of these parameters on metal uptake by
metal mixture, maximum specific uptake of 19.2 mg g1 was
B. bassiana in multimetal mixture was investigated.
observed at 100 mg L1 concentration. A decrease in biomass cou-
pled with increase in specific metal uptake is generally observed
3.4. Effect of pH and temperature on metal removal in multi metal
with increase in initial heavy metal concentration for other fungal
mixture
isolates such as A. lentulus (Mishra and Malik, 2014) and R. arrhizus
(Uslu et al., 2003). Increase in specific metal ion uptake was also
pH is an important parameter which plays a crucial role in
observed with fungus R. arrhizus when initial heavy metal concen-
metal uptake by micro-organism. pH influences the speciation of
tration was increased from 10 mg L1 to 75 mg L1 for Cu(II), Cd(II)
the heavy metal in the solution as well as the surface properties
and Pb(II) (Uslu et al., 2003). Moore et al. (2008) reported highest
of the micro-organisms. Significant decrease in metal removal
capacity (52.6%) from multi metal mixture by B. bassiana was
Table 3 observed at pH 4 as compared to the neutral pH (84.5%). At low
Bioaccumulation of Cu(II), Ni(II), Cd(II), Zn(II), Cr(VI) and multi metal mixture in pH, association of hydronium ions with the cell surface creates a
composite media by Beauveria bassiana (at 30 C, 150 rpm and 120 h). positive charge on the fungus cell wall and create repulsive force
Metal Co Xm Ct qm Uptake yield against heavy metal cations in the solution (Khalid et al., 2011).
(mg L1) (g L1) (mg L1) (mg g1) (%) Similarly, decrease in metal removal capacity (43%) was observed
Cu(II) 30 4.90 22.24 4.54 74.13 with the increase in pH up to 9. The decline in metal removal at
50 4.23 29.7 7.02 59.40 extreme pH can also be attributed to low biomass production
100 3.56 43.5 12.22 43.50 under pH stress (Fig. 3). Biomass significantly decreased at extreme
Ni(II) 30 5.01 22.5 4.49 75.00 pH (2.99 g L1 at pH 4 and 2.49 g L1 at pH) as compared to that at
50 4.23 24.9 5.89 49.80 pH 7 (4.85 g L1) in the presence of multi heavy metals. No
100 4.03 18.4 4.57 18.40
significant change in the biomass was observed in the control
Cd(II) 30 4.23 19.02 4.50 63.40 experiments at different pH (Fig. 3). Overall, multi metal removal
50 2.81 27 9.61 54.00
did not fluctuate significantly in pH ranging from 6 to 8, thereby
100 1.61 21.8 13.54 21.80
indicating the suitability of the strain form decontamination of
Zn(II) 30 5.32 20.34 3.82 67.80
wastewater used for irrigational purposes. Further, it was observed
50 4.67 23 4.93 46.00
100 3.12 38.2 12.24 38.20
that heavy metal removal was also dependent on the temperature
at which the biomass was grown. Biomass growth was observed at
Cr(VI) 30 4.82 18.34 3.80 61.13
50 3.78 22 5.82 44.00
three different temperature and it was observed that 30 C was
100 2.13 24.2 11.36 24.20 most optimum temperature for biomass production with 84.5%
Multi 30 4.85 25.36 5.23 84.53
removal. Whereas, at 20 C and 40 C, heavy metal removal per-
metal 50 3.02 23.2 7.68 46.40 centage decreased to 40.4% and 43.0%, respectively, as illustrated
100 2.12 40.7 19.20 40.70 in Fig. 4. It can be inferred that metal removal at different
X: Dried biomass; Co: Initial metal ion concentrations; Ct: metal removal after five
temperatures was dependent on biomass production. Next, to
days; qm: Specific metal ion uptake determined as the amount of metal per unit of understand and visualise the actual process of metal uptake by
dry biomass. B. bassiana, different microscopic analysis were performed.
394 D. Gola et al. / Bioresource Technology 218 (2016) 388396
Fig. 3. Biomass production in control (Xc) and multi metal (Xmm) with multi metal removal (%) by Beauveria bassiana at different initial pH (410). (Initial concentration of
heavy metal 30 mg L1; 30 C; 150 rpm).
3.5. FTIR
The FTIR spectra of fungal biomass grown in the control as well
as in the presence of heavy metal were obtained in the range of
6504000 cm1. FTIR gives the possible information on functional
group present on the cell surface and their interaction with the
heavy metals. Significant, shift in the nature of FTIR spectra bands
of fungus grown with heavy metal as compared to control were
obtained. Fig. S1 displays the numbers of absorption peaks of the
control biomass and shift in these absorption peak bands in heavy
metal containing biomass. The spectra for control fungal biomass
shows prominent peaks at 3407.0 cm1 (representing bonded
ACOOH and AOH group, primary and secondary amine and
amides, NAH stretching); 2924.7 cm1 and 2854.7 cm1(CAH
stretching); 1742.7 cm1(C@O stretching); 1643.6 cm1 (Protein
amide I band mainly C@O stretching); 1408.3 cm1 (CAH bending,
CH3); 1041.0 cm1 (CAOAC of polysaccharides) (Kaushik et al.,
2010).
However, as compared to control fungal biomass, in spectra of
fungal biomass grown in presence of heavy metal (Fig. S1BG),
peak corresponding to (representing bonded ACOOH and AOH
group, Primary and secondary amine and amides, NAH stretching);
CAH stretching; C@O stretching; CAH bending, CH3; CAOAC have
been shifted or masked. Also, in spectra of Cu(II) and Cd(II) grown
biomass, new peaks at 1460.8 cm1 and 1461.2 cm1 (representing
protein (CH2) and (CH3) bending of methyl, lipid (CH2) bending of
Fig. 4. Biomass production in control (Xc) and multi metal (Xmm) with multi metal removal (%) by Beauveria bassiana at different initial temperature (2040 C). (Initial
concentration of heavy metal 30 mg L1 and 150 rpm).
methyl), respectively, have appeared which was totally absent in
the control biomass. Amino and carboxyl group are known to bind
Cu(II) and Cd(II) (Akar and Tunali, 2005). FTIR spectra in presence
of Cu(II) and Cd(II) indicated that similar groups were involved in
binding of these heavy metals. This indicates interaction of heavy
metals with these specific functional groups present on cell sur-
face. Similarly, in spectra of biomass grown in presence of Ni(II)
new peaks at 2345.2 cm12240.6 cm1 lipid carbohydrate
mainly (CH2) and (CH2) stretching and 705.5 cm1 (representing
CAH alkene) have appeared. Whereas, in presence of Cr(VI) and
Zn(II), shift and masking of all the above peaks was observed.
The FTIR spectra obtained in present study indicated that amino
groups were involved in binding of Cr(VI) as previously observed
by Bai and Abraham (2002) with Rhizopus nigricans. The binding
of Zn(II) with phosphoryl groups was observed for Penicillium
chrysogenum (Sarret et al., 1998) correlating with the observations
obtained in the present study. New peak of carbohydrate (CAOAC)
of polysaccharide at 1357 cm11423 cm1 was observed in all the
metal treated sample. Moreover, carbohydrate (CAOAC) of
polysaccharides at (980 cm11072 cm1) which was present in
control was totally absent in presence of heavy metal. In presence
of multi metal, maximum spectral shift occurs at 3359.45 cm1
and 1074 cm1, representing protein stretching [(NAH) stretching
amide] and nucleic acid and other phosphate-containing
compounds (>P@O) stretching of phosphodiesters, respectively.
Moreover, small shifts in every wavelength as compared to control
 D. Gola et al. / Bioresource Technology 218 (2016) 388396 395

was observed in presence of multi metal system, indicating the 3.8. TEM
involvement of various functional groups.
The ultrastructural changes caused by heavy metals were
further investigated by using TEM. The cross sectional area of the
3.6. AFM
fungal cell were observed to locate the possible sites for heavy
metal accumulation. Cell structure of Beauveria bassiana under con-
AFM analyses of the fungus grown in the control condition as
trol conditions showed well defined cell membrane with intact cell
well as in the presence of heavy metal also indicate the toxicity of
and organized distribution of cytoplasm as shown in Fig. S5A. In
heavy metal. Roughness analysis is one of the parameter which indi-
presence of heavy metals, the ultrastructural changes with visible
cates the changes in the cell surface due to changes in the growing
dark spots were observed under TEM micrograph (Fig. S5BG). Min-
environment. The root mean square (rms) value corresponds to the
imum damage to the cell membrane was displayed by Cd(II) with
roughness of the sample. The rms values of the fungus in control
few dark spots present inside the cell, suggesting the accumulation
was 5.6 nm whereas in the presence of 30 mg L1 each Cd(II), Cu
of Cd(II). The bioaccumulation of Ni(II) and Cu(II) caused more
(II), Cr(II), Zn(II), Ni(II) and multi metal it was found to be 8.0 nm,
damage to the cell in comparison to Cd (II) with dense dark area,
10.1 nm, 10.7 nm, 12.8 nm, 12.8 nm and 13.2 nm, respectively
highlighting the presence of Ni(II) and Cu(II) inside the cell. While
(Fig. S2). Significant variation in the roughness values clearly indi-
in the case of Cr(VI) and Zn(II) dark spots have acquired much area
cates the bumps in the surface of fungus cell due to heavy metal
both in cell membrane and also inside the cell, suggesting the
exposure. The change in surface roughness caused by Cd(II) was
involvement of both bio-accumulation as well as bio-sorption. A
minimum as compared to other heavy metals and multi metal mix-
clear accumulation (dark dense zone) and cell damage of highest
ture. Similar trend was also observed in SEM and TEM micrographs
degree was recorded in multiple heavy metals with visible presence
of Beauveria bassiana in presence of Cd(II), suggesting less affinity
of metals both in membrane as well as inside the cell. This corre-
for Cd(II), thereby causing lesser damage in cell morphology as
lated with the results obtained with the SEM, EDX and AFM analysis
compared to the other heavy metals. The maximum roughness
(Sections 3.63.7). Similar dark and dense areas were observed both
value was observed with multi metal mixture, showing more pro-
in cell membrane and inside the Beauveria bassiana cell grown in
nounced damage and deformation of fungal hyphae, correlating
presence of Zn(II) and Pb(II) (Purchase et al., 2009).
with the observations obtained in SEM and TEM analysis Similar
increases in the roughness value of Aspergillus versicolor was
observed in presence of heavy metal (Das et al., 2008). 4. Conclusion

The aim of the present study was to examine the bioaccumula-

3.7. SEM/EDX
The SEM images show clearly distinction between the fungus
grown in the control condition (Fig. S3A) and metal stressed
condition (Fig. S3BE). Hyphae in the control condition had long
ribbon like structure with uniform shape and were packed loosely.
However, in the presence of individual heavy metals, hyphae were
short, packed densely and ribbon like structure was clearly
deformed. Relatively denser, tightly packed deformed hyphae with
shortened length were observed in the multi metal spiked media,
indicating combined toxicity of heavy metal mixtures (Fig. S3E).
The morphological changes in the hyphae can be attributed to the
toxicity response of the fungus when exposed to the heavy metals.
Similar observation were made by Mishra and Malik, (2012) where
twisting and looping of hyphae as well as formation of intertwined
hyphal strands were observed in response to the metal toxicity.
Toxic heavy metal may also bind to the surface groups of the fungus
membrane causing its integrity loss. Aggregation of fungal hyphae
in presence of heavy metal can be morphological strategy to
overcome the toxic effect of heavy metals. Aggregation leads to
the reduction in the total surface area of the fungal hyphae which
is exposed to the toxic environment (Baldrian, 2003).
Additionally, the EDX analysis of the fungus established the
presence of heavy metal on the fungus biomass as shown in
Fig. S4. The EDX graph of control biomass shows prominent peaks
of alkali and alkaline earth metal. The significant heavy metal
peaks in the EDX monograph clearly indicate the presence of the
particular heavy metal on the fungus surface. In multi metal
exposed biomass, the peaks for Cu(II), Cr(VI), Cd(II), Zn(II) and Ni
(II) were observed. Moreover, the decrease in the peak intensity
of alkali and alkaline earth metal such as Na+, K+, Ca2+ and Mg2+
in the metal exposed fungus in some cases indicates the existence
of a significant ion-exchange mechanism for cation binding (Akar
and Tunali, 2005). Most significant decrease in the Na+ ion was
depicted in fungus loaded with Cu(II), Ni(II) and Zn(II). Signal of
C, O, S and P are likely from the protein and enzyme present on
the biomass surface.
tion by B. bassiana for the individual and multi-metal ions abreast.
SEM, AFM, TEM and FTIR demonstrated the changes induced by
heavy metals and indicated specific functional groups contributing
towards metal uptake. Multi metal mixture showed highest metal
removal (84.5%) that did not fluctuate significantly in pH ranging
from 6 to 8. Hence, B. bassiana is a potential candidate for remedia-
tion of multi metals from irrigational waters due to bio-pesticidal
and metal remediation ability. Further studies on heavy metal
uptake from actual wastewater and bioreactor development are
warranted to evolve working technology.
Acknowledgements
Authors gratefully acknowledge NFBSFARA, Indian Council of
Agricultural Research (grant no. NFBSAFARA/WQ-2023/2012-13)
for funds and AB acknowledges CSIR for fellowship (09/086
(1193)/2014-EMR I). Dr. Harpal Singh (Centre for Biomedical
Engineering, IIT Delhi), Dr. Deepak varandani (Department of
Physics, IIT Delhi) and Mr. D. C. Sharma (Department of Textile
Engineering, IIT Delhi) are also acknowledged for their kind techni-
cal support in FTIR, AFM and SEM-EDX analysis, respectively.
Author gratefully acknowledge AIRF, JNU for TEM analysis.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2016.06.
096.
References
Aikel, ., Alp, T., 2009. A study on the inhibition kinetics of bioaccumulation of Cu
(II) and Ni(II) ions using Rhizopus delemar. J. Hazard. Mater. 168, 14491458.
http://dx.doi.org/10.1016/j.jhazmat.2009.03.040.
Ahmad, I., Ansari, M.I., Aqil, F., 2006. Biosorption of Ni, Cr and Cd by metal tolerant
Aspergillus niger and Penicillium sp. using single and multi-metal solution.
Indian J. Exp. Biol. 44, 7376.
396 D. Gola et al. / Bioresource Technology 218 (2016) 388396
Akar, T., Tunali, S., 2005. Biosorption performance of Botrytis cinerea fungal by-
products for removal of Cd(II) and Cu(II) ions from aqueous solutions. Miner.
Eng. 18, 10991109. http://dx.doi.org/10.1016/j.mineng.2005.03.002.
Anand, P., Isar, J., Saran, S., Saxena, R.K., 2006. Bioaccumulation of copper by
Trichoderma viride. Bioresour. Technol. 97, 10181025. http://dx.doi.org/
10.1016/j.biortech.2005.04.046.
Bai, R.S., Abraham, T.E., 2002. Studies on enhancement of Cr(VI) biosorption by
chemically modified biomass of Rhizopus nigricans. Water Res. 36, 12241236.
http://dx.doi.org/10.1016/S0043-1354(01)00330-X 2.
Baldrian, P., 2003. Interactions of heavy metals with white-rot fungi. Enzyme
Microb. Technol. 32, 7891. http://dx.doi.org/10.1016/S0141-0229(02)00245-4.
Bhattacharya, A., Dey, P., Gola, D., Mishra, A., Malik, A., Patel, N., 2015. Assessment of
Yamuna and associated drains used for irrigation in rural and peri-urban
settings of Delhi NCR. Environ. Monit. Assess. 187, 4146. http://dx.doi.org/
10.1007/s10661-014-4146-2.
Bulgariu, D., Bulgariu, L., 2012. Equilibrium and kinetics studies of heavy metal ions
biosorption on green algae waste biomass. Bioresour. Technol. 103, 489493.
http://dx.doi.org/10.1016/j.biortech.2011.10.016.
Chang, J.S., Huang, J.C., 1998. Selective adsorption/recovery of Pb, Cu, and Cd with
multiple fixed beds containing immobilized bacterial biomass. Biotechnol.
Progr. 14 (5), 735741.
Das, S.K., Mukherjee, M., Guha, A.K., 2008. Interaction of chromium with resistant
strain Aspergillus versicolor: investigation with atomic force microscopy and
other physical studies. Langmuir 24, 86438650. http://dx.doi.org/10.1021/
la800958u.
Dnmez, G., Aksu, Z., 2001. Bioaccumulation of copper(ii) and nickel(ii) by the non-
adapted and adapted growing Candida sp. Water Res. 35, 14251434. http://dx.
doi.org/10.1016/S0043-1354(00)00394-8.
Dursun, A.Y., Uslu, G., Cuci, Y., Aksu, Z., 2003. Bioaccumulation of copper(II), lead(II)
and chromium(VI) by growing Aspergillus niger. Process Biochem. 38, 1647
1651. http://dx.doi.org/10.1016/S0032-9592(02)00075-4.
Gola, D., Chauhan, N., Malik, A., Ahammad, Z.S., 2016. Bioremediation approach for
handling multiple metal contamination. In: Das, Surajit, Dash, Hirak R. (Eds.),
Handbook of Metal-Microbe Interactions and Bioremediation. Taylor & Francis
(CRC Press), ISBN 9781498762427 (in press).
Guo, H., Luo, S., Chen, L., Xiao, X., Xi, Q., Wei, W., Zeng, G., Liu, C., Wan, Y., Chen, J.,
He, Y., 2010. Bioremediation of heavy metals by growing hyperaccumulaor
endophytic bacterium Bacillus sp. L14. Bioresour. Technol. 101, 85998605.
http://dx.doi.org/10.1016/j.biortech.2010.06.085.
Gupta, S.K., Chabukdhara, M., Kumar, P., Singh, J., Bux, F., 2014. Evaluation of
ecological risk of metal contamination in river Gomti, India: a biomonitoring
approach. Ecotoxicol. Environ. Saf. 110, 4955. http://dx.doi.org/10.1016/j.
ecoenv.2014.08.008.
Kameo, S., Iwahashi, H., Kojima, Y., Satoh, H., 2000. Induction of metallothioneins in
the heavy metal resistant fungus Beauveria bassiana exposed to copper or
cadmium. Analusis 28, 382385. http://dx.doi.org/10.1051/
analusis:2000280382.
Kaushik, A., Sharma, H.R., Jain, S., Dawra, J., Kaushik, C.P., 2010. Pesticide pollution of
River Ghaggar in Haryana, India. Environ. Monit. Assess. 160, 6169. http://dx.
doi.org/10.1007/s10661-008-0657-z.
Khalid, A., Hussein, Sedky, H.A., Hassan, J.H.J., 2011. Potential capacity of Beauveria
bassiana and Metarhizium anisopliae in the biosorption of Cd2+ and Pb2+. J. Gen.
Appl. Microbiol. 57 (6), 347355, 57, T publish.
Khodabakhsh, F., Nazeri, S., Amoozegar, M.A., Khodakaramian, G., 2011. Isolation of
a moderately halophilic bacterium resistant to some toxic metals from Aran &
Bidgol salt lake and its phylogenetic characterization by 16s rDNA gene. Feyz J.
Kashan Univ. Med. Sci.
Li, C., Xu, Y., Jiang, W., Dong, X., Wang, D., Liu, B., 2013. Effect of NaCl on the heavy
metal tolerance and bioaccumulation of Zygosaccharomyces rouxii and
Saccharomyces cerevisiae. Bioresour. Technol. 143, 4652. http://dx.doi.org/
10.1016/j.biortech.2013.05.114.
Lpez Errasqun, E., Vzquez, C., 2003. Tolerance and uptake of heavy metals by
Trichoderma atroviride isolated from sludge. Chemosphere 50, 137143. http://
dx.doi.org/10.1016/S0045-6535(02)00485-X.
Machado, M.D., Soares, E.V., Soares, H.M.V.M., 2010. Removal of heavy metals using
a brewers yeast strain of Saccharomyces cerevisiae: chemical speciation as a tool
in the prediction and improving of treatment efficiency of real electroplating
effluents. J. Hazard. Mater. 180, 347353. http://dx.doi.org/10.1016/j.
jhazmat.2010.04.037.
Mishra, A., Malik, A., 2012. Simultaneous bioaccumulation of multiple metals from
electroplating effluent using Aspergillus lentulus. Water Res. 46, 49914998.
http://dx.doi.org/10.1016/j.watres.2012.06.035.
Mishra, A., Malik, A., 2014. Novel fungal consortium for bioremediation of metals
and dyes from mixed waste stream. Bioresour. Technol. 171, 217226. http://
dx.doi.org/10.1016/j.biortech.2014.08.047.
Mishra, S., Singh, S.N., Pande, V., 2014. Bacteria induced degradation of fluoranthene in
minimal salt medium mediated by catabolic enzymes in vitro condition. Bioresour.
Technol. 164, 299308. http://dx.doi.org/10.1016/j.biortech.2014.04.076.
Moore, B.A., Duncan, J.R., Burgess, J.E., 2008. Fungal bioaccumulation of copper,
nickel, gold and platinum. Miner. Eng. 21, 5560. http://dx.doi.org/10.1016/j.
mineng.2007.08.005.
Nguyen, T.A.H., Ngo, H.H., Guo, W.S., Zhang, J., Liang, S., Yue, Q.Y., Li, Q., Nguyen, T.V.,
2013. Applicability of agricultural waste and by-products for adsorptive
removal of heavy metals from wastewater. Bioresour. Technol. 148, 574585.
http://dx.doi.org/10.1016/j.biortech.2013.08.124.
Pakshirajan, K., Swaminathan, T., 2009. Biosorption of lead, copper, and cadmium by
Phanerochaete chrysosporium in ternary metal mixtures: statistical analysis of
individual and interaction effects. Appl. Biochem. Biotechnol. 158, 457469.
http://dx.doi.org/10.1007/s12010-008-8374-1.
Pan, R., Cao, L., Zhang, R., 2009. Combined effects of Cu, Cd, Pb, and Zn on the growth
and uptake of consortium of Cu-resistant Penicillium sp. A1 and Cd-resistant
Fusarium sp. A19. J. Hazard. Mater. 171, 761766. http://dx.doi.org/10.1016/j.
jhazmat.2009.06.080.
Purchase, D., Scholes, L.N.L., Revitt, D.M., Shutes, R.B.E., 2009. Effects of temperature
on metal tolerance and the accumulation of Zn and Pb by metal-tolerant fungi
isolated from urban runoff treatment wetlands. J. Appl. Microbiol. 106, 1163
1174. http://dx.doi.org/10.1111/j.1365-2672.2008.04082.x.
Sag, Y., Akcael, B., Kutsal, T., 2002. Ternary biosorption equilibria of chromium(VI),
copper(II), and cadmium(II) on Rhizopus arrhizus. Sep. Sci. Technol. 37, 279309.
http://dx.doi.org/10.1081/SS-120000789.
Sarret, G., Manceau, A., Spadini, L., Roux, J.C., Hazemann, J.L., Soldo, Y., Eybert-
Brard, L., Menthonnex, J.J., 1998. Structural determination of Zn and Pb binding
sites in Penicillium chrysogenum cell walls by EXAFS spectroscopy. Environ. Sci.
Technol. 32, 16481655. http://dx.doi.org/10.1021/es9709684.
Scholes, L.N.L., Shutes, R.B.E., Revitt, D.M., Purchase, D., Forshaw, M., 1999. The
removal of urban pollutants by constructed wetlands during wet weather.
Water Sci. Technol. 40, 333340. http://dx.doi.org/10.1016/S0273-1223(99)
00467-9.
Shah, M.P., 2014. Eco-friendly treatment of acid red by an application of
Pseudomonas spp. Int. J. Environ. Bioremediation Biodegrad. 2, 6268. http://
dx.doi.org/10.12691/ijebb-2-2-3.
Uslu, G., Dursun, A.Y., Ekiz, H.I., Aksu, Z., 2003. The effect of Cd (II), Pb (II) and Cu (II)
ions on the growth and bioaccumulation properties of Rhizopus arrhizus. Process
Biochem. 39, 105110. http://dx.doi.org/10.1016/S0032-9592(03)00032-3.
Yadav, A.K., Kumar, N., Sreekrishnan, T.R., Satya, S., Bishnoi, N.R., 2010. Removal of
chromium and nickel from aqueous solution in constructed wetland: mass
balance, adsorptiondesorption and FTIR study. Chem. Eng. J. 160, 122128.
http://dx.doi.org/10.1016/j.cej.2010.03.019.