Geomicrobiology Journal

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2+
Comparative Analysis of Mechanisms of Cd
2+
and Ni Biosorption by Living and Nonliving
Mucoromycote sp. XLC

Wei Zhu, Xingjian Xu, Lu Xia, Qiaoyun Huang & Wenli Chen

To cite this article: Wei Zhu, Xingjian Xu, Lu Xia, Qiaoyun Huang & Wenli Chen (2016)
2+ 2+
Comparative Analysis of Mechanisms of Cd and Ni Biosorption by Living and
Nonliving Mucoromycote sp. XLC, Geomicrobiology Journal, 33:3-4, 274-282, DOI:
10.1080/01490451.2015.1052114

To link to this article: http://dx.doi.org/10.1080/01490451.2015.1052114

Published online: 25 Feb 2016.

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 GEOMICROBIOLOGY JOURNAL
2016, VOL. 33, NOS. 3–4, 274–282
http://dx.doi.org/10.1080/01490451.2015.1052114

Comparative Analysis of Mechanisms of Cd2C and Ni2C Biosorption by Living

and Nonliving Mucoromycote sp. XLC
Wei Zhua, Xingjian Xua,b, Lu Xiaa, Qiaoyun Huanga,c, and Wenli Chena
a
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China; bNortheast Institute of Geography and
Agroecology, Chinese Academy of Sciences, Changchun, China; cKey Laboratory of Arable Land Conservation (Middle and Lower Reaches of Yangtze
River), Ministry of Agriculture, College of Resources and Environment, Huazhong Agricultural University, Wuhan, China

ABSTRACT ARTICLE HISTORY

The biosorption of cadmium (Cd2C) and nickel (Ni2C) using living and nonliving biomass of Mucoromycote Received March 2015
Downloaded by [University of California, San Diego] at 00:59 01 March 2016

sp. XLC as a biosorbent was investigated in this study. The optimum conditions were established with Accepted May 2015
batch biosorption experiments. The data from equilibrium experiments showed that all kinds of KEYWORDS
biosorbents fitted the Langmuir model. For the living and nonliving biosorbent, the predicted maximum Biosorption; cadmium;
Cd2C uptake capacity was 79.65 mg g¡1 and 56.51 mg g¡1, while the maximum Ni2C uptake capacity was Mucoromycote sp.; nickel
51.26 mg g¡1 and 44.32 mg g¡1, respectively. Acid digestion results indicated that intracellular metal ions
might be the main reason for the metal uptake capacity difference between living and nonliving
biosorbents. FTIR analysis and potentiometric titration indicated freeze dehydration-autoclaving treatment
of nonliving biomass slightly changed the surface structure of the cell wall and the functional groups
involved in biosorption were carboxylic, phosphoryl, amine and hydroxyl groups. Our results suggest the
living biosorbent had a second biosorption process by intracellularly accumulating metal ions, hence
possessing a higher Cd2Cor Ni2C uptake capacity than nonliving biosorbent.

Introduction
Heavy metal pollution has been a serious environmental prob-
lem caused by rapid industrialization, which is a particular con-
cern in developing countries (Celik and Demirbas 2005).
Wastewater containing heavy metals discharge into natural
water-body systems, which may be used for agriculture pur-
poses, thus resulting in the accumulation of toxic substances in
soils and crops. Unfortunately, heavy metals in environment
are not biodegradable and tend to concentrate in living organ-
isms. In this case, these hazardous materials will constitute a
potential risk for ecosystems and human health (Demirbas
2008).
Cd2C and Ni2C often exist in industrial wastewaters, and are
mentioned in the contamination list of 13 metals proposed by
the United States Environmental Protection Agency (USEPA)
(Aaseth and Norseth 1986). The toxicity of Ni2C to living
organisms is usually effected on enzymes, because Ni2C has a
high affinity for ligands containing oxygen, nitrogen and sulfur.
Excess Ni2C is a potential human carcinogen and may cause
cancer of the lungs, nose and bone (Beliles 1979; Ramos et al.
2002). In addition, nickel is also associated with reproductive
problems and birth defects (Xu et al. 2008). Cadmium is
another extremely toxic pollutant originating from metal plat-
ing, metallurgical alloying, mining, ceramics and other indus-
trial operations (Davis et al. 2003; Khlifi et al. 2014). The
toxicity of this metal is usually observed as renal dysfunction,
hypertension, lung damage and teratogenic effects (Aziz et al.
2014).
Conventional methods for cleansing heavy metal ions from
wastewaters include chemical treatment, ion exchange, carbon
adsorption, coprecipitation/adsorption and membrane filtra-
tion (Hajialigol et al. 2006), are more or less ineffective, expen-
sive and difficult to implement in developing countries
(Rangsayatorn et al. 2004; Sari and Tuzen 2009), especially
when the metals in wastewater are in the range 1–100 mg L¡1
(Pan et al. 2005). Therefore, a simple, cost-effective and eco-
friendly treatment strategy for removing metal ions is of great
importance. The ability of biological materials to absorb metal
ions has been causing great interest in developing an efficient,
clean and cheap technology even at metal concentrations as
low as 1 mg L¡1 (Chong and Volesky 1995; Joshi and Sahu
2014).
Various kinds of bacteria, fungi, algae, mosses, macrophytes
and higher plants have been examined for removing heavy met-
als and seem to have the potential for practical application.
However, the use of living or nonliving biomass for the removal
of metal ions still remains a debatable question.
Living biomass, such as yeast cells may have a higher metal-
removing capacity due to the transport of metal ions across the
cell membrane (Volesky and May-Phillips 1995). On the other
hand, the advantages of using nonliving biomass for industrial

CONTACT Wenli Chen wlchen@mail.hzaui.edu.cn State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China;
Qiaoyun Huang qyhuang@mail.hzau.edu.cn State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
Wei Zhu and Xingjian Xu contributed equally to this article.
Color versions for one or more of the figures in the article can be found online at www.tandfonline.com/ugmb.
© 2016 Taylor & Francis Group, LLC

applications such as ease of storage, insensitivity to metal toxic-
ity and nutrient supply has been stressed (Gadd 1990). Brady et
al. (1994) illustrated that hot alkali yeast treatment could mod-
erately decrease Cu2C accumulation when compared with
native yeast cells. Other researchers reported that dead biomass
showed a lower Sr2C (Avery and Tobin 1992) or Pb2C (Suh and
Kim 2000) uptake than the respective living-biosorbents. Cop-
per uptake by living S. cerevisiae yeast cells is biphasic, which is
consisting of an initial rapid surface binding of copper ions, fol-
lowed by a slower intracellular uptake of copper (de Rome and
Gadd 1987; Huang et al. 1990). The mechanisms used by living
biomass for the removal of heavy metal ions seem quite differ-
ent from that of nonliving biomass.
The aim of the current study was to compare Cd2C and Ni2C
biosorption in living and nonliving biomass of Mucoromycote
sp. XLC. The influence of different parameters on the biosorp-
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tion capacity was investigated and different equilibrium models
were applied to describe the biosorption process. Potentiomet-
ric titration and FTIR analysis were also conducted to identify
the probable functional groups on the fungal surface, thus dem-
onstrating the differential mechanism of Cd2C and Ni2C uptake
by living and nonliving biomass.
Materials and methods
Biosorbent preparation
Mucoromycote sp. XLC was isolated from chicken manure
compost (purchased from Wuhan Biological Engineering Com-
pany, China) by incubation on potato dextrose agar (PDA)
plates at 28 C for 4 days to produce adequate spores. Spores
were collected in 2-mL sterile distilled water, aseptically trans-
ferred to 500 mL-flasks containing 200 mL minimal medium
(MM: 5 g (NH4)2SO4, 1.5 g KH2PO4, 0.6 g MgSO4, 0.6 g CaCl2,
0.005 g FeSO4·7H2O, 0.002 g CoCl2, and 20 g glucose per liter
of medium) and incubated with continuously shaking at
180 rpm and 28 C for 4 days. Two kinds of flasks were used in
this experiment.
The conventional Erlenmeyer flask was used for nonliving
biosorbent preparation. Specially designed flasks, which have
three raised corners in the bottom, were used for collection of
living biosorbent. After appropriate incubation, mycelia were
harvested by centrifugation at 4000 £ g for 5 min and washed
thrice with deionized water. For "nonliving biosorbent," the
mycelia collected from the conventional flasks were freeze
dehydrated, then autoclaved at 121 C for 20 min and freeze
dehydrated again. The freeze dehydrated mycelium was ground
to obtain homogeneous particles. The living biosorbent col-
lected from the specially designed flasks was stored at 4 C
before for use. Part of the living biomass were taken and dried
at 80 C until it attained a constant weight, in order to calculate
the water content of the living biosorbent.
Chemicals
Stock solutions (1000 mg L¡1) of Ni2C, Cd2C, K2C, Ca2C, Na2C,
Mg2C, Cu 2C and Cr3C were prepared by dissolving an exact
amount of analytical grade NiCl2, Cd(NO3)2, KNO3, CaCl2,
NaCl, MgCl2, CuCl2, and Cr(NO3)3 in deionized water,
GEOMICROBIOLOGY JOURNAL 275
respectively. Other concentrations were obtained by dilution.
Fresh dilutions were used for each biosorption experiment.
Batch biosorption studies
The effects of solution pH, contact time, initial metal concen-
tration and the biosorbent dose on biosorption ability were
investigated. For all experimental treatments, a fixed volume of
metal solution (20 mL) was introduced with the desired bio-
sorbent dose in the 50-mL Erlenmeyer flasks. Flasks were
shaken constantly at 150 rpm and 28 C. The effect of initial pH
on biosorption capacity was conducted at a pH range of 2.0 to
6.0, which was adjusted using 0.1 M NaOH and 0.1 M HCl.
Various initial metal solutions with different concentrations
(0–500 mg L¡1) of Cd2C and Ni2C were prepared from metal
stock solutions.
Contact time (0–240 min) and biosorbent dose (0.5, 1.0, 2.5,
and 5.0 g L¡1) were also evaluated in the current study. The
amounts of living and nonliving biosorbents used in these
experiments were all calculated on a dry weight biomass basis.
After the desired contact time, the metal solution was filtered
through 0.22-mm Millipore membrane filters and stored at
28 C before analytical measurements. All treatments were car-
ried out in triplicate. Residual metal ions in solution were mea-
sured using an atomic absorption spectrophotometer (F-240,
Varian). The removal of metal ions was calculated through the
equation described by Xu et al. (2012).
Equilibrium experiments and equilibrium models
For each type of biosorbent, equilibrium experiments were con-
ducted under the optimum conditions determined by the batch
biosorption experiments. Briefly, living and nonliving biosorb-
ents were added to the solutions of Cd2C and Ni2C at concen-
trations ranging from 0 to 500 mg L¡1, agitated at 150 rpm and
28 C for 4 h.
Two popular adsorption models such as Langmuir and
Freundlich models are often used to evaluate isotherm data and
to determine the type of biosorption (Khambhaty et al. 2009).
The Langmuir isotherm model is appropriate for the mono-
layer type sorption where all sites on the surface of the sorbent
have the same affinity and assume no migration of metal ions
in the surface sites. The Langmuir equation is expressed as fol-
lows (Langmuir 1916):
qmax Keq Ce
qe D [1]
1 C Keq Ce
1
RL D [2]
1 C Keq C0
qmax represents the theoretical maximum biosorption ability of
the biosorbent (mg g¡1) and Keq (L mg¡1) is the equilibrium
constant and a measure for the intensity of biosorption. Ce (mg
L¡1) and qe (mg g¡1) are the metal ion concentrations at equi-
librium and the biosorption ability at any time, respectively.
Another essential constant RL reflects the biosorption process
and is classified into unfavorable (RL > 1), linear (RL D 1),
favorable (0 < RL < 1) or irreversible (RL D 0).
 276 W. ZHU ET AL.
The Freundlich isotherm model describes adsorption to het-
erogeneous surfaces (Gadd 2009), which is mathematically
expressed as follows (Freundlich 1906):
n
1 40 min, and then back-titrated with 0.0978 M NaOH to a pH
qe D KF Ce f [3]
Ce and qe are the same as mentioned previously, and KF and
nf are the Freundlich model constants.
Acidic digestion of biosorbents
The living and nonliving biosorbents were collected after bio-
sorption and then freeze dehydrated. The freeze-dried biosorb-
ents were washed with metal eluant (50 mM Tris-HCl, 10 mM
EDTA-Na2) three times and then rinsed with deionized water
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three times to remove the metals adsorbed on the surface.
Thereafter, the biosorbents were freeze dehydrated again.
Approximately 0.1–0.2 g dried biosorbents treated with desorp-
tion agent and two zeolite beads were wetted with 1 mL deion-
ized water. Then 5 mL of H2SO4 was subsequently introduced
into the digestion tube (an acid-washed tube that was
completely dried), and then incubated at room temperature
overnight. HClO4 (0.5 mL) was transferred into the tube and
digested at 180 C for 30 min followed by a temperature of
300 C for at least 5 h until complete digestion of the biosorb-
ent. After cooling to room temperature, the digested solution in
the tube was transferred into a 25-mL volumetric flask and
appropriate deionized water was added to the 25-mL scale line.
After filtration using 0.22-mm membrane filters, the obtained
solution was used for determining metal concentrations by
AAS. Blanks were treated in the same way.
FTIR spectroscopy
Infrared spectra of living and nonliving biosorbents after expo-
sure to cadmium, nickel and deionized water were measured.
Living and nonliving biosorbents (0.5 g L¡1 dry weight bio-
mass) were suspended in the metal solutions at their normal
initial pH and a concentration of 500 mg L¡1. The biosorbents
treated with deionized water were set up as a control treatment.
Each type of biosorbent was freeze dehydrated, mixed with KBr
in the ratio of 1:100, and ground to homogenous particles. The
samples were analyzed by a Fourier transform infrared spectro-
scope (FTIR, Nicolet 5700, Thermo) in the range of 4000–400
cm¡1, which provides a preliminary characterization of the
main functional groups associated with biosorption on the cell
wall. The background was automatically subtracted from the
sample spectra.
Potentiometric titration
Potentiometric titration experiments were performed to iden-
tify and determine the types and concentrations of the acidic
sites on the fungal cell wall that may be responsible for metal
biosorption using an automatic potentiometric titrator
(Metrohm Titrator 836). Living and nonliving biosorbents
(2.053–2.906 g L¡1 in dry weight) were suspended in 0.1 M
HNO3 and the suspensions were kept under a N2 atmosphere
at 25 C. The deionized water used in this experiment must be
boiled to remove dissolved CO2.
Each suspension was titrated to the low pH of approximately
2.5 by adding aliquots of 0.1008 M HCl, equilibrated for
of 10.0. The titration experiments in this study were performed
to a maximum pH of 10.0, which does not cause loss of cell via-
bility or cell damage. In addition, the desired starting pH of 2.5
can avoid excessive disruption of the cell wall functional group
structure (Borrok et al. 2004). Blank titration used 0.1M KNO3
without biosorbent. Titrations were performed in triplicate and
Protofit 2.1 was used to analyze the experimental data.
Statistical analysis
All data analyses were performed using an SPSS 13.0. All plots
were made using SigmaPlot (version 10.0). The data were eval-
uated by ANOVA with significance set at p < 0.05.
Results and discussion
Effect of pH
Initial pH of the metal solution has been demonstrated to be
one of the important parameters in governing sorption effi-
ciency of various sorbents for heavy metals (Aksu 2005). The
acidity of the metal solution not only affects the surface charge
of the biosorbent but also influences metal solubility. In the
current study, once the pH was above 6.0 and 7.0, it would
cause the precipitation of Cd2C and Ni2C, respectively. Thus,
the range of initial pH values for Cd2C solutions was adjusted
to pH 2.0–6.0. For the solutions of Ni2C, the pH was in the
range of pH 2.0–7.0. As shown in Figure 1a, it was obvious that
pH had a significant influence on biosorption by living and
nonliving biosorbents of Cd2C and Ni2C.
The maximum biosorption capacity of living and nonliving
biomass for Cd2C was at pH 5.0 and 4.0, respectively. For Ni2C,
the maximum biosorption ability peaked at pH 6.0 and 5.0,
respectively. In addition, the biosorption capacity of all the
tested biosorbents increased steadily with an increase of pH
from 2 to the respective favorable pH, and then decreased as
the pH increased to the desired maximum pH value for Cd2C
and Ni2C in this study. Kiran and Akar (2005) indicated more
functional groups with negative charges such as carboxyl,
amine or hydroxyl become exposed with a subsequent increase
of attraction sites to positively charged ions when pH was above
5.0, thus enhancing biosorption ability. In this study, the lower
Ni2C biosorption ability could be explained by competition
between HC and Ni2C for anionic sites at low pH.
With increasing pH, the HC decreases and some groups
become to be electronegative to bind Ni2C on the surface of
biosorbents. Moreover, the maximum adsorption ability of liv-
ing biosorbent was higher than nonliving biosorbent. This may
be because such metal ions could be accumulated or trapped in
inside the fungal biomass through metabolism dependent pro-
cesses or such as ion transportation. The difference in favorable
pH values for biosorption of Cd2C or Ni2C by living and nonliv-
ing biosorbents might be attributed to changes in the

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Figure 1. Effect of (a) pH, (b) contact time, (c) initial metal concentration and (d) biomass dose on the biosorption of Cd2C and Ni2C by living and nonliving cells of
Mucoromycote sp. XLC, respectively. Error bars represents standard deviations of the data collected in the parallel treatments.
biomacromolecular conformation of the nonliving cell walls
caused by the high temperature pasteurization (Srinath et al.
2002)
Effect of contact time
The effects of contact time on the biosorption equilibrium in
metal solutions at their normal pH are presented in Figure 1b.
The results show that the reduction of Cd2C in solution
occurred sharply within 15 min, followed by a slow increase
until equilibration at 45 min by the nonliving biomass and at
120 min by the living biomass. No significant difference in bio-
sorption capacity was determined beyond the equilibrium time
(p > 0.05). Similar results for the biosorption for Ni2C were
observed in the current study, but with an equilibrium time of
60 min for the nonliving biomass and 180 min for the living
biomass.
Once the biosorption processes reached an equilibrated
state, there was no obvious difference in biosorption capacity
with increasing contact time (p > 0.05). The difference in bio-
sorption capacity between living and nonliving biomass was
mainly due to a more complex process existing for the removal
of metal ions by the living biomass rather than the nonliving
biomass. In brief, the first step for metal removal by the living
biosorbent was similar to the nonliving biosorbent. In the first
stage, the biosorption is always rapid and considered to be a
spontaneous process with no energy consumed. The second
phase might be a process with the metal ions accumulating
GEOMICROBIOLOGY JOURNAL 277
inside the cells, which only occurred with living cell biosorption
(Volesky and May-Phillips 1995). Similar results were reported
for the biosorption of Cd2C, Cr6C, Fe3C and Ni2C by E. coli
from aqueous solutions (Quintelas et al. 2009).
Effect of initial metal concentration
As depicted in Figure 1c, biosorption ability of all the tested
biosorbents increased with the increase of initial Cd2C or Ni2C
concentrations from 0 to 500 mg L¡1. The increasing trends in
biosorption capacity were not dependent on the metal species
and biomass types. However, the maximum biosorption ability
of living biosorbent for each tested metal was always higher
than that of the nonliving biosorbent. The maximum biosorp-
tion ability of the nonliving and living biosorbents achieved at
an initial Cd2C concentration of 300 mg L¡1 and 400 mg L¡1
was 25.65 mg g¡1 and 51.85 mg g¡1, respectively.
For Ni2C, biosorption capacity of the nonliving and living
biosorbents equilibrated at 300 mg L¡1 and 500 mg L¡1 with
24.54 mg g¡1 and 31.09 mg g¡1, respectively. Higher concentra-
tions of each metal above the equilibrium concentration only
slightly stimulated biosorption capacity of each type of bio-
mass. Because the available binding sites for the adsorption
process were limited, the excess of metal ions could not result,
therefore, in higher biosorption ability (Xu et al. 2012). How-
ever, a second process, metal uptake accompanied with energy
consumption might be involved in living cell biosorption to
enhance the biosorption capacity with an increase of metal
 278 W. ZHU ET AL.

were available, leading to increasing metal removal efficiency.

In addition, no significant difference in maximum metal
removal was found by the introduction of biomass doses
between 2.5 and 5.0 g L¡1 (p > 0.05). This result suggested that
redundant biosorbents were not helpful in biosorption at a cer-
tain concentration of heavy metals. It can be explained that bio-
mass aggregation occurred at higher biosorbent concentration,
causing a decrease in effective surface binding area of the bio-
sorbents for biosorption. Therefore, the optimum biosorbent
dose of all the tested biomass was selected as 0.5 g L¡1 for the
following experiments.

Biosorption isotherms
The data obtained from the current study were fitted for non-
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linear regression analysis and the isotherm plots are presented

Figure 2. Langmuir and Freundlich fitting plots of biosorption of (a) Cd2C and (b)
Ni2C onto living and nonliving cells of Mucoromycote sp. XLC, respectively. (sym-
bols: experimental data; lines: model prediction).
in Figures 2a and 2b. The parameters of the Langmuir model
and Freundlich model are displayed in Table 1.
The predicted qmax of Cd2C biosorption capacity of living
and nonliving biosorbent was 79.65 mg g¡1 and 56.51 mg g¡1,
respectively. Although the qmax value for Ni2C biosorption
capacity was 51.26 mg g¡1 and 44.32 mg g¡1 for living and
nonliving biosorbent, respectively. The affinity constant (Keq)
of living biosorbent was higher than that of the nonliving bio-
sorbents for Cd2C or Ni2C biosorption. Another factor RL of liv-
ing biosorbent was lower than that of the nonliving biosorbent.
These results indicated that biosorption for Cd2C or Ni2C by
the living biosorbent was more favorable than for the nonliving
one. In the Freundlich model, the constant n between 1-10
reflects a favorable adsorption trend (Noreen et al. 2010), the
larger value implies a stronger interaction between the biosorb-
ent and metal ions. As listed in Table 1, the R2 value of the two
biosorbents with the Langmuir model were slight higher than
with the Freundlich model, so therefore the Langmuir isotherm
was more adequate to describe the biosorption data.

concentrations. Hence, the biosorption ability of living biosorb-

ent was higher than the nonliving biosorbent.
Effect of biosorbent dose
Previous studies showed that biosorbent dose is also an impor-
tant factor in affecting biosorption (Li et al. 2010; Sun et al.
2011). The results obtained in this study illustrated that the
removal efficiency was increased with the biosorbent dose
increasing, but the biosorption ability decreased gradually
(Figure 1d). With an increase of biosorbent, more binding sites
Acidic digestion of biosorbents
Table 2 showed the results of the biosorption ability and the
intracellular metal concentrations of the saturated biosorbents
after acid digesting. These results indicated the possibility of
transporting the metal ions into cells through metabolic activi-
ties involved in metal uptake by the living biosorbent. In addi-
tion, intracellular metal ions should account for the
biosorption capacity difference between living and nonliving
Mucoromycote sp. XLC.

Table 1. Constants simulated with Langmuir and Freundlich models for Cd2C and Ni2C biosorption using living and nonliving Mucoromycote sp. XLC as biosorbents.
Langmuir model Freundlich model

Cadmium Nickel Cadmium Nickel

Biosorbents Keq(L mg¡1) qmax(mg g¡1) R2 RL Keq(L mg¡1) qmax(mg g¡1) R2 RL nf KF(L g¡1) R2 nf KF(L ¢ g¡1) R2

Living 0.0088 79.65 0.9944 0.2355 0.0047 51.26 0.9918 0.2275 1.944 2.6452 0.9713 1.8165 1.227 0.9745
Nonliving 0.0024 56.51 0.9919 0.8547 0.0039 44.32 0.9913 0.5306 1.4892 0.5249 0.9838 1.6529 0.681 0.9795
 GEOMICROBIOLOGY JOURNAL 279

Table 2. The biosorption ability of living and nonliving Mucoromycote sp. XLC and -CH2. The weak band observed at 1740 cm¡1 might be
the intracellular concentration of Cd2C and Ni2C after saturated biosorption. CDO stretching in unconjugated ketone, carbonyl and ali-
Biosorption ability Intracellular metal phatic groups. The 1652 cm¡1 peak of native living biosorb-
(mg g¡1) concentration (mg g¡1) ent and the 1643 cm¡1 peak of native nonliving biosorbent
Biosorbents Cadmium Nickel Cadmium Nickel were due to the stretching vibration of CDO and C N
(amide I) groups of proteins. The peak observed at 1546
Living 51.76 § 0.88 36.27 § 1.11 18.57 § 1.95 8.91 § 1.03 cm¡1 was attributed to N-H bending and C-N stretching
Nonliving 29.11 § 1.69 25.98 § 0.22 1.04 § 0.78 0.83 § 0.44
vibrations (amide II) of the peptides. The weak peak around
1460 cm¡1 might be assigned to C-H bending in benzene
ring. The peak arising at 1410 cm¡1 was the stretching of
FTIR analysis CDO from carboxylic groups (amide III). The band peaked
at 1380 cm¡1 was the stretching vibration of -CH3.
The FTIR spectrum of the raw biomass, Cd2C or Ni2C-loaded Strong peaks around 1245 cm¡1 and 1078 cm¡1 represented
living and nonliving biosorbents are shown in Figures 3a and C O C stretching and C N stretching vibration of amine
3b and the functional groups probably involved in biosorption groups or C-O deformation in alcohols, respectively. An obvi-
€
were analyzed according to previous studies (Ozdemir et al. ous peak at 1154 cm¡1 was CDO stretching in aromatic acid
Downloaded by [University of California, San Diego] at 00:59 01 March 2016

2009; Parikh and Chorover 2005; Xu et al. 2008; Yee et al.
2004). The spectrum of native living and nonliving biosorbents
showed broad and strong bands at 3433.86 cm¡1 and 3437.78
cm¡1, respectively, indicating the stretching vibrations of
hydroxyl (-OH) or amine (-NH) groups.
The peaks at the region near 2924 cm¡1 (living) and
2960 cm¡1 (nonliving) were possibly attributed to the asym-
metric stretching vibration of -CH2 or -CH3 groups of the
fatty acid on the cell membrane. The band that peaked at
2855 cm¡1 could be assigned as the symmetric stretching of
groups. The peak at 1030 cm¡1 might be due to P-O vibration
of the phosphoryl groups. On analysis, the FTIR spectra
between the raw living biosorbent and the raw nonliving bio-
sorbent were nearly the same except for the obvious differences
at 1643 cm¡1, 1459 cm¡1 and 1236 cm¡1, respectively. How-
ever, the peaks ranging from 1155 cm¡1 to 1380 cm¡1 indicated
that the autoclaving process might have destroyed the structure
of the aromatic series. The region ranging from 500 to 800
cm¡1 is called the "fingerprint" and represents phosphate or
sulfur functional groups

Figure 3. FT-IR spectra of (a)living cells and (b)nonliving cells of Mucoromycote sp. XLC loaded with and without Cd2C or Ni2C.
 280 W. ZHU ET AL.

On the whole, the spectra of biosorbents loaded with Cd2C

or Ni2C presented slight changes in comparison with the raw
biosorbents. For the Cd2C loaded living biosorbent, the peaks
at 3433.86 cm¡1, 1463.43 cm¡1, 1408.07 cm¡1, 1379.52 cm¡1,
and 1250.63 cm¡1 shifted to 3438.38 cm¡1, 1459.41 cm¡1,
1413.76 cm¡1, 1384.71 cm¡1, and 1245.63 cm¡1, respectively
(Figure 3a).
Although no strong shifts existed, the shifts of detected peaks
showed that -OH or -NH groups, C H, -CH3 and -C6H5 groups
might participate in Cd2C biosorption. For the nonliving biosorb-
ent, the spectral analysis before and after Cd2C biosorption indi-
cated that the -OH or -NH groups, -CH3 and C H were
involved in the Cd2C biosorption process. In addition, a substan-
tial increase of adsorption intensity occurred at 1384.71 cm¡1 for
the living biosorbent and 1383.84 cm¡1 for the nonliving biosorb-
ent after Cd2C biosorption, from which it can be deduced that
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polysaccharides might also play an important role in Cd2C bio-

sorption. Some other groups such as -CH2, C-N, CDO and
P O might also participate in the Cd2C biosorption process.
The spectral data of living and nonliving biosorbents loaded
with Ni2C showed fewer changes than that loaded with Cd2C
(Figure 3b). In case of the living biosorbent, the major func-
tional groups were -OH or -NH groups, -CH3, -COOH and
-C6H5 groups. In addition, only -OH or -NH groups exhibited
obvious adsorption intensity after Ni2C biosorption to the non-
living biosorbent. In conclusion, the kinds of functional groups
involved in metal biosorption were similar between the living
and nonliving biosorbents, but there were more functional
groups participating in the biosorption of Cd2C than Ni2C.
Potentiometric titrations
The potentiometric titration curves of living and nonliving
Mucoromycote sp. XLC are presented in Figures 4a and 4b as
the concentration of deprotonated sites was standardized per
mass of dry weight of biosorbent (mol g¡1) versus pH. The
data was fitted used ProtoFit 2.1 with the Non-Electrostatic
Model (NEM) being used to fit the acid base titration data to
quantify the active surface functional groups on the tested bio-
mass (Fein et al. 2005; Turner and Fein 2006).
The biosorbent suspensions exhibited a significant proton
buffering behavior as revealed by the observation of pH
changes. The buffering capacity was due to the functional
groups on the surface of biosorbent, which could consume the
added acid by accepting protons and the added base by donat-
ing protons. During the whole titration process, no evidence of
saturation was observed with respect to proton adsorption. If
the functional groups were protonated completely, it would
cause the slope of the titration curve to match that of the blank
titration under low pH conditions. As shown in Figures 4a and
4b, all the titration curves of the biosorbent suspension had
steeper slopes than the blank titration. This result showed the
Figure 4. Potentiometric titration data for cell suspensions of (a)living Mucoromy-
cote sp. XLC cells and (b) nonliving Mucoromycote sp. XLC cells compared with
0.1M KNO3 as a blank.
functional groups were not fully protonated even at the lowest
pH of 2.5. The buffering capacity of nonliving biosorbent was
higher than that of the living biosorbent (Figures 4a and 4b). In
addition, only slight variability existed in the three replicates of
each biosorbent.
Table 3 summarized the results from the ProtoFit program
with NEM based on four sites. The same pH range and the
same model of two tested biosorbents made it possible to com-
pare their pKa values and the functional site concentrations.
The results illustrated that the functional groups were classified
into four main kinds, but except for the most acidic site, the
constant pKa values of the other three sites of the two tested
biosorbents were similar. The obtained pKa values could be
assigned to carboxylic groups (2.0–6.0), phosphoryl groups
(5.6–7.2), amine groups (9.0–11.0), and hydroxyl groups (8.0–
12.0), respectively (Leone et al. 2007).
The calculated concentrations of surface functional groups
are also shown in Table 3. Site densities were provided in the
range of 10¡5 moles per gram of biosorbent. The concentra-
tions of carboxylic groups (C1) and amine/hydroxyl groups

Table 3. Deprotonation constants and surface site concentration of living and nonliving Mucoromycote sp. XLC as calculated using Protofit 2.1.
Biosorbents pK1 pK2 pK3 pK4 C1(10¡4 mol g¡1) C2(10¡4 mol g¡1) C3(10¡4 mol g¡1) C4(10¡4 mol g¡1) Csum(10¡4 mol g¡1)

Living 5.00 § 0.17 6.56 § 0.09 8.69 § 0.07 10.38 § 0.27 2.17 § 0.28 3.56 § 0.37 9.31 § 1.25 9.87 § 0.74 24.91
Nonliving 4.31 § 0.28 6.78 § 0.04 8.42 § 0.29 10.04 § 0.38 3.26 § 0.09 13.54 § 1.39 4.09 § 0.79 7.90 § 1.43 28.79

(C4) were nearly the same. However, the density of the
hydroxyl groups of the living biosorbent was slightly higher
than the nonliving biosorbent. The concentration of the phos-
phoric groups was markedly less than the nonliving biosorbent.
The total buffering capacity (the total site concentration) for
the living biosorbent was 24.91 £ 10¡5 mol g¡1, which was a
little lower than that for the nonliving biosorbent (28.79 £
10¡5 mol g¡1). Moreover, the order of biosorption capacity
agreed with the curves described. The inflection spots at pH
8–9 (Figure 4a) and pH 6–7 (Figure 4b) should be explained by
the dominant concentration variances of phosphoric groups of
nonliving biosorbent and hydroxyl groups of living biosorbent,
respectively.
The difference in buffering capacity between the living and
nonliving Mucoromycote sp. XLC evaluated with the potentio-
metric titration data indicated that the freeze dehydration-
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autoclaving treatment might lead to some changes in fungal
cell wall structure, allowing more active phosphoric groups to
participate in proton binding. The specific surface area of the
biosorbent might be another reason for causing the difference.
The nonliving biosorbent had been ground to obtain homoge-
nous particles while the living fungal cells always gathered as
mycelial pellets and therefore some functional sites might be
covered, reducing the opportunity for proton binding.
Combining the results of acidic digestion (Table 2), FT-IR
analysis (Figures 3a and 3b) and potentiometric titrations
(Figures 4a and 4b), only a slight discrepancy of functional groups
on the living and nonliving Mucoromycote sp. XLC was deter-
mined. The active mode of transporting metal ions caused the
differentiation in biosorption ability of the tested biomass. More-
over, either the living or nonliving biosorbent had a higher Cd2C
biosorption ability than Ni2C. This phenomenon might be related
to the kinds of functional groups which have a specific affinity to
Cd2C on the surface of Mucoromycote sp. XLC. In addition, the
fungal strain XLC was isolated from a Cd-contaminated soil, pos-
sibly making it accumulate more intracellular Cd2C than Ni2C.
Conclusions
In this study, living and nonliving cells of Mucoromycote sp.
XLC displayed significant abilities for Cd2C and Ni2C sorption.
Optimum pH relied on metal species and the type of biomass.
The metabolic activity of the tested strain obviously affected
equilibrium metal concentration. The amount of optimum bio-
mass was not dependent on the biosorbent and metals. The
data obtained from equilibrium experiments fitted to the Lang-
muir model. The biosorption mechanism was mainly related to
the interaction between metal ions and the acidic sites (func-
tional groups) on the surface of the cell walls, which were con-
firmed by FTIR analysis and potentiometric titration. Our
results suggest that strain XLC is a promising biosorbent for
removing Cd2C and Ni2C ions from wastewater. However, the
recycling of the biosorbent should be considered for further
practical applications.
Funding
This work was supported by National High Technology Research and
Development Program of China (“863” Program, 2012AA101402), the
GEOMICROBIOLOGY JOURNAL 281
National Natural Science of Foundation of China (41230854) and the Pro-
gram for Changjiang Scholars and Innovative Research Team in University
of China (IRT1247).
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