Biochemical Engineering Journal 34 (2007) 131–135

Bioaccumulation of chromium(VI), copper(II) and nickel(II)

ions by growing Rhizopus arrhizus
B. Preetha ∗ , T. Viruthagiri
Department of Technology, Annamalai University, Annamalai Nagar, Chidambaram 608002, Tamil Nadu, India
Received 1 October 2006; received in revised form 25 November 2006; accepted 27 November 2006

Abstract
The metal resistance capacity and metal ion accumulation capacity during the growth of Rhizopus arrhizus and the inhibition kinetics of heavy
metals, namely chromium, copper and nickel were studied in a batch reactor. A maximum percentage uptake yield of 93.84, 95.52 and 61.44%
were found for 25 mg/l of initial metal ion concentrations of chromium, copper and nickel, respectively. A maximum biomass of 20.17 g/l was
obtained at 20 g/l of initial dextrose concentration and in absence of heavy metals. The inhibition was found to be a competitive inhibition for
the bioaccumulation of chromium. Lineweaver–Burk plot, Aiba model and Bazua and Wilke models were used to study the inhibition kinetics of
bioaccumulation of heavy metals chromium, copper and nickel using R. arrhizus and the model parameters were evaluated using experimental data.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Bioaccumulation; Heavy metals; Rhizopus arrhizus; Growth curve; Inhibition kinetics; Metal–microbe interaction

1. Introduction tion by living cells. In inactivated biomass (biosorption), the

The removal of heavy metal ions from industrial wastewater
is a problem of increasing concern that has been mostly solved
by chemical and physical methods of treatment. However, these
procedures have significant disadvantages, such as incomplete
metal removal, high reagent or energy requirements, or gener-
ation of toxic sludge or other waste products and are generally
very expensive when the contaminant concentrations are in the
range 10–100 mg/l [1]. Chromium is a toxic metal of widespread
use in many industries such as metal plating facilities, mining
operations and tanneries. Besides chromium, copper and nickel
are also common and serious environmental pollutants. They are
frequently encountered together in industrial wastewaters, e.g.
electroplating, electronics wire manufacturing, oil refineries and
copper sulphate manufacture [2–4]. Therefore, there is a need for
the development of low cost, easily available materials, which
could adsorb toxic heavy metals from wastewater. Microor-
ganisms are potent bioremediators, removing heavy metals via
active or passive uptake mechanisms. Metal accumulative bio-
processes generally fall into one of two categories, biosorptive
uptake by non-living, non-growing biomass and bioaccumula-
microorganisms usually sequester metal through surface bond-
ing only; with active biomass (bioaccumulation), metals are
concentrated through a combination of surface reactions, intra-
and extracellular precipitation, and intra- and extracellular com-
plexation reactions [5]. The phenomenon of metal accumulation
in the microorganism will enhance the metal toxicity and in turn
reduces the growth of microorganisms. Using growing cultures
in bioremoval could avoid the need for a separate biomass pro-
duction process such as cultivation, harvesting, drying, etc., but
the requirements to maintain cell growth. The effectiveness of
these processes is usually dependent on the parameters such
as temperature, toxicity, oxygen level and availability of nutri-
ents, etc. If the problem of metal toxicity to the growing cell is
overcome by the use of metal-resistant organisms, the continu-
ally self-replenishing system can be left to run continuously for
extended periods [6].
The objective of the present study was to investigate the effect
of heavy metal ions on the growth and bioaccumulation prop-
erties of Rhizopus arrhizus as a function of initial metal ion
concentration in batch experiments.
2. Experimental and mathematical models

∗ Corresponding author. Tel.: +91 4144 239479. R. arrhizus (MTCC 2233), a filamentous fungus was obtained
E-mail address: preethapar@yahoo.co.in (B. Preetha). from the Institute of Microbial Technology, Chandigarh, India.

1369-703X/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2006.11.022
132 B. Preetha, T. Viruthagiri / Biochemical Engineering Journal 34 (2007) 131–135
Nomenclature
Ks saturation constant (g/l)
P product concentration (mg/l)
Pcrit critical product concentration (mg/l)
S substrate concentration (g/l)
Greek symbols
μ specific growth rate (h−1 )
μmax maximum specific growth rate (h−1 )
The microorganism was grown aerobically in agitated enrich-
ment media containing following composition: potato extract
(200 g/l) and dextrose (20 g/l) at 25 ◦ C. The pH of the medium
was adjusted to 5.3 with dilute sulphuric acid.
Metal ion solutions of chromium(VI), nickel(II) and cop-
per(II) were prepared by dissolving their respective salts,
namely potassium dichromate (K2 Cr2 O7 ), nickel sulphate
(NiSO4 ·6H2 O) and copper sulphate (CuSO4 ·5H2 O), respec-
tively, in double distilled water.
The factors affecting the growth and metal ion uptake rate
of R. arrhizus were examined in 500 ml Erlenmeyer flasks with
100 ml of accumulation medium containing the respective metal
ion solutions either chromium or nickel or copper ions with vary-
ing concentrations from 25 to 100 mg/l. The pH of the solution
was adjusted to the desired value by using dilute sulphuric acid
and autoclaved at 121 ◦ C for 15 min. The sterilized accumula-
tion medium was mixed with 100 ml of sterilized enrichment
media containing potato extract and dextrose with desired pH.
Five millilitres of inoculum was added to the above medium and
the culture was grown at 25 ◦ C and aeration was maintained by
shaking at 200 rpm for 72 h. This shaking frequency supplied
the culture with enough oxygen to attain logarithmic growth.
The samples were drawn at pre-determined time intervals from
a flask containing the same culture grown at identical conditions
for measuring the residual metal ion concentrations and biomass
concentrations.
Experiments were also carried out to study the growth of R.
arrhizus in the varying dextrose concentration from 5 to 25 g/l at
pH 5.6 at 25 ◦ C in the absence of metals under identical condi-
tions. The samples were drawn at pre-determined time intervals
from a flask containing the same culture grown at identical con-
ditions for measuring the dextrose concentration and biomass
concentrations.
The residual chromium, copper and nickel ion concentra-
tion in the medium were determined spectrophotometrically at
540, 457 and 445 nm using diphenylcarbazide, neocuproine and
dimethylglyoxime as the complexing agent [7]. The dextrose
concentrations were also measured spectrophotometrically at
510 nm by dinitrosalicylic acid method [8]. The dry weight of
the R. arrhizus was determined after the organism had been dried
at 40 ◦ C for 2 h.
Development of suitable mathematical models which would
describe the metal inhibition and growth kinetics is very use-
ful in designing the bioaccumulation systems using live cells.
Several mathematical models have been proposed to quantify
the inhibitory effects of the end product on cellular growth.
Since metal ions cannot be degraded into other products, but can
undergo changes in valency and/or convert into organo-metallic
compounds, an attempt has been made to characterize the metal
inhibition by end product inhibition.
2.1. Lineweaver–Burk plot
The well-known Monod expression for the growth kinetics
does not represent the inhibitory effects of toxic end products.
Lineweaver–Burk plot is the linearized form of Monod equation
and is used to evaluate the kinetic parameters of bioaccumulation
system accurately [9].
1 Ks 1 1
= + (1)
μ μmax S μmax
where μ is the specific growth rate (h−1 ), μmax the maximum
specific growth rate (h−1 ), S the substrate concentration (g/l) and
Ks is the saturation constant (g/l).
2.2. Aiba model
Though growth kinetics for most of the systems is well rep-
resented by Monod model, it fails to account for the inhibition
effects. The inhibition effect mainly depends on the factors such
as concentration of the metal ions, the tolerance capacity of the
microorganisms and the operating conditions of the bioaccumu-
lation process. Metal ions are considered to be the products in
bioaccumulation of heavy metals by microorganism, its concen-
tration on the microorganism increases with respect to time as
bioaccumulation proceeds. The toxic effect of the metal ions ulti-
mately affects the growth kinetics. As metal ion concentration
increases specific growth rate decreases and the Monod equation
must include a term for product inhibition [10].
μ = f (S, P) (2)
where P is the product concentration (mg/l). Aiba’s exponen-
tial model has been widely used to predict the bioaccumulation
kinetics in presence of inhibition due to an exponential term e−kp
which takes care of the product inhibition on growth kinetics of
R. arrhizus. Aiba’s exponential model, though has been widely
used to analyze product inhibition, fails to give the critical value
of inhibitory product concentration [9].
μmax S −kp
μ= e (3)
Ks + S
For given cell mass and substrate concentration, Eq. (3) pre-
dicts the specific growth rate more accurately for all product
concentration.
2.3. Bazua and Wilke model
Bazua and Wilke’s model developed for non-competitive
inhibition can be used to characterize the metal inhibition
kinetics of R. arrhizus and to find the inhibitory product concen-
tration. The specific growth rate is affected by metal ions even
 B. Preetha, T. Viruthagiri / Biochemical Engineering Journal 34 (2007) 131–135
at very low concentrations. The growth rate of microorganism is
inhibited by metal ions in a non-competitive manner. The term
(1 − P/Pcrit )0.5 is used to account for the inhibition of metals on
the growth of R. arrhizus. It is also used to find the Pcrit , the crit-
ical concentration of product below which there is no inhibition
[11–13].
 
μmax S P 0.5
μ= 1−
Ks + S Pcrit
where Pcrit is the critical product concentration (mg/l). The
kinetic data obtained in the batch growth studies were used to
evaluate the kinetic model parameters of the Lineweaver–Burk
plot, Aiba model and Bazua and Wilke models for chromium,
copper and nickel, respectively.
3. Results and discussion
Metal contaminated environments can be treated by microor-
ganism, which can accumulate heavy metal ions in their
cells during growth either internally or externally. The results
obtained from the effect of initial chromium ion concentra-
tions on the growth of R. arrhizus were given as, the maximum
biomass obtained (dry weight), the maximum bioaccumulated
chromium ion concentration at the end of growth, the maximum
specific uptake of chromium metal ion and the percentage uptake
yield.
Fig. 1 shows the effect of chromium ion concentration on the
growth of R. arrhizus. Increasing the concentration of chromium
ions in the growth medium caused an increase in lag period.
This shows that the chromium ion bioaccumulation was mainly
dependent on metabolic activity. From the results, a lag period
of 12 h, log period of 44 h and a stationary period of 60 h were
observed. A maximum biomass of 20.17 g/l was obtained at
20 g/l of initial dextrose concentration.
Fig. 2 shows the inhibition kinetics of growth of R. arrhizus
by chromium. When initial metal ion concentration increases
from 25 to 100 mg/l the uptake yield of chromium was found to
decrease from 93.84 to 35%, respectively, whereas the maximum
specific uptake of chromium increases from 1.58 to 5.36 mg/g up
to 75 mg/l and thereafter it decreases to 4.55 mg/g with a further
increase in initial metal ion concentration to 100 mg/l. A maxi-
mum biomass concentration of 14.87 g/l was obtained at 25 mg/l
Fig. 1. Effect of dextrose concentration on the growth of Rhizopus arrhizus.
133
(4)
Fig. 2. Inhibition kinetics of growth of Rhizopus arrhizus by chromium.
initial chromium concentration. This clearly indicates that as
metal ion concentration increases within the cells, the growth
rate and the maximum biomass yield decreases and hence the
surface area available for sorption decreases thereby a reduction
in specific uptake of chromium was observed and indicated in
Fig. 2. During the growth period as metal accumulates in the
organism, the metabolic activity of microorganism decreases.
At low metal ion concentrations, the ratio of sorptive surface to
the total metal ions available is high and nearly all metal ions in
solution can be bound and removed.
The kinetic data obtained in the batch growth studies were
used to evaluate the kinetic parameters under no inhibition con-
ditions and the results were given in Fig. 3. The studies on the
effect of dextrose concentration on the specific growth rate of
organism reveals that the specific growth rate increases signifi-
cantly with increase in the substrate concentration. The results
show that the optimum concentration was 20 g/l since it gave a
maximum specific growth rate as indicated in Fig. 3. The satu-
ration constant (Ks ) and maximum specific growth rate (μmax )
were found to be 7.69 g/l and 0.085 h−1 , respectively, from
Fig. 3.
Bioaccumulation of chromium with 20 g/l of initial dextrose
concentration was studied in presence and in absence of heavy
metals and the concentration profile of biomass and dextrose
was given in Fig. 4, which then used to find out the type of
inhibition using Lineweaver–Burk plot. It was observed from
the Lineweaver–Burk plot, the inhibition may be a competitive
Fig. 3. Effect of dextrose concentration on specific growth rate of Rhizopus
arrhizus Ks = 7.69 g/l and μmax = 0.085 h−1 .
134 B. Preetha, T. Viruthagiri / Biochemical Engineering Journal 34 (2007) 131–135

Fig. 4. Inhibition kinetics for the growth of Rhizopus arrhizus in presence and Fig. 5. Product inhibition modeling on the bioaccumulation of chromium.
in absence of chromium ions (50 mg/l) with 20 g/l dextrose concentration.

one since the μmax value remains constant at 0.037 h−1 while
the Ks value increases from 0.170 to 0.217 g/l for 50 mg/l of
chromium.
The studies on the effect of chromium, nickel and copper ion
concentration on specific growth rate of the organism reveals
that the specific growth rate decreases substantially with an
increase in the initial metal ion concentration. This indicates
that the growth rate of microorganism appears to be inhibited
by the presence of heavy metals. The experimental kinetic data
obtained in the batch growth studies of R. arrhizus were used to
evaluate the model parameters in the inhibition model, namely
Aiba model and Bazua and Wilke model. The model parameters
Fig. 6. Product inhibition modeling on the bioaccumulation of copper.
were evaluated by Gauss–Jacobian method using Matlab 6.5.
The parameter values obtained were then used to simulate the
model to predict the growth and bioaccumulation capacity of R.
arrhizus. Table 1 shows the calculated model parameters for the
inhibition kinetics of chromium, copper and nickel on the growth
of R. arrhizus and also indicates the critical metal ion concen-
tration, below which there is no inhibition. Figs. 5–7 show the
experimental and predicted specific growth rate data for Aiba
and Bazua and Wilke model used for the removal of chromium,
copper and nickel, respectively. The results indicate that the Aiba
model predicts closely the inhibition effect of chromium, cop-
per and nickel and represents the competitive inhibition very
well. Bazua and Wilke model does not represent the inhibi-
tion effect of chromium, copper and nickel on the growth of
R. arrhizus accurately which indicates the tolerance limit of
microorganisms. In the present study, the metal inhibition on Fig. 7. Product inhibition modeling on the bioaccumulation of nickel.

Table 1
Calculated model parameters for the inhibition kinetics of chromium, copper and nickel on the growth of Rhizopus arrhizus
Model Metal ion Model parameters

K Pcrit (mg/l) μmax (h−1 ) Ks (g/l)

Aiba et al. (1968) Chromium 0.007 – 0.032 0.568

Copper 0.018 – 0.045 0.445
Nickel 0.003 – 0.033 2.13
Bazua and Wilke (1977) Chromium – 321 0.038 3.56
Copper – 117 0.059 6.53
Nickel – 704 0.041 5.53

Experimentally determined values: μmax = 0.085 h−1 ; Ks = 7.69 g/l.

 B. Preetha, T. Viruthagiri / Biochemical Engineering Journal 34 (2007) 131–135
growth kinetics was found to be competitive inhibition resulting
in greater reduction in percentage uptake yield due to the compe-
tition between the substrate (dextrose) and metal ions. The Bazua
and Wilke model was originally developed for non-competitive
inhibition and hence a large deviation was found between the
experimental and predicted values. The critical metal ion con-
centration from Bazua and Wilke model was found to be 321,
117 and 704 mg/l for the bioaccumulation of chromium, copper
and nickel below which there is no inhibition occurs. As metal
ion concentration increases the inhibition effect on the growth
of R. arrhizus increases and the deviation between the experi-
mental and predicted values also increases particularly it is more
for copper.
4. Conclusions
The aim of this work was to find the bioaccumulation capac-
ity of R. arrhizus for the selective removal of chromium, copper
and nickel ions during the growth. In the present study, as the
initial metal ion concentration increases the specific growth rate
of R. arrhizus decreases for chromium, copper and nickel. A
maximum biomass of 20.17 g/l was obtained at 20 g/l of ini-
tial dextrose concentration and in absence of heavy metals.
The kinetic parameters of the growth kinetics were found to be
Ks = 7.69 g/l and μmax = 0.085 h−1 . The inhibition was found to
be a competitive inhibition for the bioaccumulation of chromium
since the μmax was same for other experiments in presence of
metal ions. Aiba model was found to represent accurately the
inhibition kinetics. Bazua and Wilke model showed some devi-
ations from the experimental results for chromium, copper and
nickel, respectively.
135
References
[1] Y. Sag, T. Kutsal, Copper(II) and nickel(II) adsorption by Rhizopus arrhizus
in batch stirred reactors in series, Chem. Eng. J. 58 (1995) 265–273.
[2] N. Fiol, I. Villaescusa, M. Martinez, N. Miralles, J. Poch, J. Serarols,
Biosorption of chromium(VI) using low cost sorbents, Environ. Chem.
Lett. 1 (2003) 135–139.
[3] A. Magyarosy, R.D. Laidlaw, R. Kilaas, C. Echer, D.S. Clark, J.D. Keasling,
Nickel accumulation and nickel oxalate precipitation by Aspergillus niger,
Appl. Microbiol. Biotechnol. 59 (2002) 388–392.
[4] Y. Sag, I. Atacoglu, T. Kutsal, Simultaneous biosorption of chromium(VI)
and copper(II) on Rhizopus arrhizus in packed bed reactor: application of
the competitive Freundlich model, Sep. Sci. Technol. 34 (1999) 3155–3171.
[5] G. Donmez, Z. Aksu, The effect of copper(II) ions on the growth and
bioaccumulation properties of some yeasts, Process Biochem. 35 (1999)
135–142.
[6] A.Y. Dursun, G. Uslu, O. Tepe, Y. Cuci, H.I. Ekiz, A comparative inves-
tigation on the bioaccumulation of heavy metal ions by growing Rhizopus
arrhizus and Aspergillus niger, Biochem. Eng. J. 15 (2003) 87–92.
[7] APHA, AWWA, Standard Methods for the Examination of Water and
Wastewater, 19th ed., Washington, DC, 1994.
[8] S. Sadasivam, A. Manickam, Biochemical Methods, second ed., 1996, pp.
6–7.
[9] K. Han, O. Levenspeil, Extended Monod kinetics for substrate, product and
cell inhibition, Biotechnol. Bioeng. 32 (1988) 430–437.
[10] J.H.T. Luong, Kinetics of ethanol inhibition in alcohol fermentation,
Biotechnol. Bioeng. 27 (1985) 280–285.
[11] S. Aiba, M. Shoda, M. Nagatani, Kinetics of product inhibition in alcohol
fermentation, Biotechnol. Bioeng. 10 (1968) 845–864.
[12] C.D. Bazua, C.R. Wilke, Ethanol effects on the kinetics of a continuous
fermentation with Saccharomyces cerevisiae, Biotehnol. Bioeng. 7 (1977)
105–118.
[13] K. Pakshirajan, T. Swaminathan, Studies on inhibitory effects of copper(II)
and lead(II) on the growth of Phanerochaete chrysosporium, in: Proceed-
ings of 54th Annual Session of Indian Institute of Chemical Engineers,
2001, TSM-201.