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Trakya University Journal of Natural Sciences, 22(1): 67-76, 2021
ISSN 2147-0294, e-ISSN 2528-9691
DOI: 10.23902/trkjnat.817451
OPEN ACCESS
© Copyright 2021 Mersin & Açıkel
Research Article
PRODUCTION OF Candida BIOMASSES FOR HEAVY METAL REMOVAL
FROM WASTEWATERS
Gülşah MERSİN1*, Ünsal AÇIKEL2
1 Uşak University, Technology Transfer Office, Project Support Unit, Rectorate Ground Floor Office No: 106, 64000, Uşak,
TURKEY
2 Cumhuriyet University, Faculty of Engineering, Chemical Engineering Department, 58140, Sivas, TURKEY
Cite this article as:
Mersin G., Açıkel Ü. 2021. Production of Candida biomasses for heavy metal removal from wastewaters. Trakya Univ J Nat Sci, 22(1): 67-76, DOI:
10.23902/trkjnat.817451
Received: 28 October 2020, Accepted: 06 April 2021, Online First: 11 April 2021, Published: 15 April 2021
Edited by:
Bülent Yorulmaz
*Corresponding Author:
Gülşah Mersin
gulsah.mersin@gmail.com
ORCID iDs of the authors:
GM. orcid.org/0000-0002-2852-6114
ÜA. orcid.org/0000-0003-4969-8502
Key words:
Bioaccumulation
Heavy metal cations
Wastewater
Candida
Biomass
Abstract: Yeasts can accumulate heavy metals and grow in acidic media. In the present
study, it was shown that Candida yeasts in an aqueous solution accumulate single Cu(II) and
Ni(II) cations. The effect of heavy metal ions on the specific growth rate of biomasses and
the uptake of metal ions during the growth phase was investigated in a batch system.
Bioaccumulation efficiency decreased with increasing metal ion concentrations at constant
sucrose concentrations. Both the specific growth rate and the biomass concentration were
more inhibited in the bioaccumulation media containing Ni(II) ions singly as compared with
the bioaccumulation media containing Cu(II) ions singly. The maximum specific growth rate
and the saturation constant of yeasts were examined with a double-reciprocal form of Monod
equation. Metal uptake performance decreased from 81.68% to 46.28% with increasing Ni(II)
concentration from 25 mg/L to 250 mg/L for Candida lipolytica. Candida biomasses may be
an alternative way of removal of heavy metals from wastewaters and may constitute a sample
to produce new biomass. The study showed that Candida yeasts can be used as economical
biomass due to their metal resistance and efficient production.
Özet: Mayalar, asidik ortamda büyüyebilir ve ağır metalleri biriktirebilir. Bu çalışma,
Candida türü mayaların sulu çözeltilerden tekli Cu(II) ve Ni(II) katyonlarını biriktirdiğini
göstermiştir. Ağır metal iyonlarının, biyokütlelerin spesifik büyüme hızı ve büyüme periyodu
boyunca metal iyonlarını giderimi üzerindeki etkisi, bir kesikli sistemde araştırılmıştır. Sabit
sakaroz derişiminde, metal iyonu derişimi arttıkça, biyobirikim verimi azalmıştır. Hem
spesifik büyüme hızı hem de biyokütle konsantrasyonu, tek başına Cu(II) iyonları içeren
biyoakümülasyon ortamına kıyasla Ni(II) iyonları içeren biyoakümülasyon ortamında daha
fazla inhibe edilmiştir. Mayaların maksimum özgül büyüme hızı ve doygunluk sabiti, Monod
denkleminin çift-karşılıklı formu ile incelenmiştir. Candida lipolytica’nın metal giderim
performansı Ni(II) derişiminin 25 mg/L’den 250 mg/L'ye çıkmasıyla % 81,68'den % 46,28'e
düşmüştür. Candida biyokütleleri, ağır metallerin atık sulardan gideriminde alternatif bir yol
olabilir ve yeni biyokütle üretimi için bir örnek oluşturabilir. Bu çalışma, Candida
mayalarının, metal direnci ve verimli üretimleri nedeniyle ekonomik biyokütle olarak
kullanılabileceğini göstermektedir.
Introduction
Clean water plays a vital role in living organisms.
Industrial activities cause water contamination due to
chemical, physical and biological components in water
bodies. One of the important sources of water
contamination is heavy metals. The presence of heavy
metals in the environment may cause significant hazards
to both animals and humans. Even in trace amounts, heavy
metals play a vital role in human metabolic systems, and
high concentrations of trace elements are toxic, and they
cause physiological and neurological hazards
(Tchounwou et al. 2012). Several methods are used for
the treatment of wastewater effluents. These methods
include chemical precipitation, ion exchange, adsorption,
membrane filtration, reverse osmosis, solvent extraction
etc. (Wolowiec et al. 2019). Some adsorbents such as
clay, zeolite, fly ash, agro wastes, and chitin have been
reported as low-cost for the removal of contaminations
from aqueous solutions. Biomass can be derived from
both vegetables and animals, either living or dead, and is
used as an adsorbent to efficiently remove heavy metals
from wastewaters. Biomasses have some advantages such
as high efficiency, minimal sludge formation,
regeneration, and no additional supplementary of
nutrients (Tripathi & Ranjan 2015). Yeasts which are used
68 G. Mersin & Ü. Açıkel
in the enzymatic industry and medicine can survive in a
medium containing low or high concentrations of heavy
metals (Cottet et al. 2020). One of the most important
microbial source for biosorption of heavy metals is
Candida species (Luna et al. 2016), which were shown to
play an important role in the accumulation of metal ions
(Honfi et al. 2016, Luk et al. 2017). Metal uptake capacity
of Candida species under various experimental conditions
depends on the metal type and the yeast species itself
(Legorreta-Castañeda et al. 2020). Bioaccumulation, which
contains some processes as complex formation, ion
transfer, adsorption, and chelation is applied to eliminate
toxic effects of heavy metals as a cheap, efficient, and green
technology (Redha 2020, Fadel et al. 2017). The
biosorption of metals is affected by several factors such as
pH, temperature, concentration, type of biomass, contact
time, and type of metal ions in solution. Bioaccumulation
based on the accumulation of metals in living
microorganisms is metabolism dependent (Açıkel & Alp
2009). The bioaccumulation of heavy metals by living cells
contains two stages. The first step is very fast due to surface
adsorption carried out on the surface of the microorganism
with physical adsorption and ion exchange. The second
step is the intracellular metal uptake stage which occurs
slower due to the metabolic activity of microorganisms
(Podder & Majumder 2019). Bioaccumulation has been
investigated in many studies for the removal of heavy
metals from wastewaters. Cd(II) removal by Candida
tropicalis, Cu(II) removal by C. utilis, Pb(II) removal by C.
albicans can be given as examples (Gönen & Aksu 2008,
Baysal et al. 2009, Rehman & Anjum 2011). In the
bioaccumulation process, high concentrations of heavy
metals may interact with microorganisms which would
result in prolonged lag time and reduced growth rate.
Therefore, microbial growth kinetics are affected by heavy
metals. Nickel is a trace element necessary for microbial
growth, but it may cause oxidative stress and disruption of
the cell membrane when in higher concentrations, (Fashola
et al. 2016). Cu(II) is one of the most stable metals and
shows a high affinity for metalloproteins in cells (Waldron
& Robinson 2009). Some microorganisms use Cu as a
catalyzer for electron transfer reactions in cell metabolism.
Microorganisms have different metal-binding proteins due
to their nature (Dupont et al. 2011). Mathematical
description of the growth kinetics can be explained by the
Monod equation (Şengör et al. 2009), which is widely used
to describe the empirical microbial growth of
microorganisms as a simple model (1).
Where µmax is the maximum growth rate when there is
enough substrate supplied to the cell and the value
exceeds the limiting substrate concentration, S>Ks. The
constant Ks is the saturation constant or half of the velocity
constant and is equal to the concentration of the rate-
limiting substrate when the specific growth rate is equal
to one-half of the maximum specific growth rate (Monod
1949, Liu 2007). Microorganism cell consists of an outer
cover called a cell wall and contains a variety of
functional sites such as amines, phosphates, sulfates,
phenols, and hydroxyls with the ability for adsorption of
metal ions (Javanbakht et al. 2013, Cottet et al. 2020).
Metal ion adsorption by microorganisms is calculated by
the mass balance equation (2).
Where qe is metal ion uptake per unit mass of biomass
at equilibrium (mg/g biomass), Ce is the metal ion
concentration in solution at equilibrium (mg/L), Co is the
initial metal ion concentration in solution (mg/L), V is
volume of the initial metal ion’s solution (L), and m is the
mass of biomass (g) (Zha et al. 2020).
In this study, bioaccumulation, and growth properties
of Candida biomasses for the uptake of Cu(II) and Ni(II)
ions were investigated as a function of molasses sucrose
and metal ion concentrations. Molasses sucrose was used
as the main carbon source. The inhibition effects of metal
ions on specific growth rates were examined. The results
showed that Candida species can be used as a biosource
for efficient removal of heavy metals from aqueous
solutions at low costs.
Materials and Methods
Candida membranafiens (C. membranafiens-ATCC®
201377™), C. utilis (C. utilis-ATCC® 9950), C.
tropicalis (C. tropicalis-ATCC® 13803™) and C.
lipolytica (C. lipolytica-ATCC® 9733™) were obtained
from the Biology Department of Ankara University.
(NH4)2SO4 and K2PO4 were purchased from Sigma-
Aldrich Company. Molasses sucrose was supplied from a
sugar factory in Ankara (Turkey). Molasses consisting of
47-48% sugar was used as the sole carbon source for the
growth of the microorganisms. Total sugars constitute of
the system included approximately 50% (w/w) of
molasses, ash 11% (w/w), and total nitrogen compounds
7-8% (w/w). Non-sugar part of molasses contained
minerals and trace elements such as K+, Na+, Ca2+, Mg2+,
Fe2+, Cl−, SO4−, PO4−, NO3− and metal oxides (as ferric
0.4-2.7%) (Açıkel & Alp 2009).
Microorganism growth and bioaccumulation media
Yeasts were grown in aqueous media containing (1-10
g/L) molasses sucrose, (1 g/L) (NH4)2SO4 and (1 g/L)
K2PO4 at 25 °C (pH: 4.0). The growth and cultivation
media were sterilized in an autoclave operating at 121°C
at 0.99 bar for 15 minutes. Subcultures were grown for 4
days at a rotating speed of 150 rpm. 1 g/L Cu(II) and
Ni(II) stock solutions were prepared by diluting Cu
(NO3)2.3H2O and Ni (NO3)2.6H2O in distilled water. The
pH of the working solutions was adjusted to desired value
by adding 0.1 N NaOH and HNO3 (pH: 4.0). The yeasts
were adapted to the metal ions in culture medium by
exposing them to single Cu(II) and Ni(II) ions during the
growth phase to increase their metal resistance. The
resistance to Cu(II) and Ni(II) ions was investigated as
functions of initial metal ion and molasses sucrose
Biomass production of Candida species 69
Trakya Univ J Nat Sci, 22(1): 67-76, 2021
concentrations and the yeasts were adapted to higher
metal ion concentrations after the first inoculation. Each
yeast was adapted to each metal ion in its culture medium.
Adapted yeasts were obtained from subcultures with
different concentrations of metal ions in the range of 25-
250 mg/L in varying concentrations from 1 g/L to 20 g/L
for molasses. Yeast cultures which were resistant to 25
mg/L Cu(II) and Ni(II) ions at 10 g/L molasses sucrose
concentration was used for further inoculation. 1 mL
culture medium was used to inoculate the next culture
medium containing 50 mg/L Cu(II) and Ni(II) ions at the
same molasses sucrose concentration when growth
culture reached to the exponential growth phase.
Adaptation experiments by Candida species were carried
out in 250 mL flask with 100 mL working volume.
Analytical procedure
5 mL samples were centrifuged at 3000xg for 5 min
and the supernatant fluid was analyzed for metal ions. The
precipitated cells were used for determination of the dry
weight of the biomass and the biomass concentration.
Yeast pellets were dried until constant mass at 60°C for
24 h. The amount of total metal ions was calculated from
the calibration graph. Microorganism concentration was
measured at 360 nm using a calibration curve relating the
wet weight of the biomass to the dry weight of the biomass
at 25°C. Residual metal concentrations were measured at
460 nm and 340 nm for Cu(II) and Ni(II), respectively by
using Sodium diethyldithiocarbamate as the complexing
agent (Sandell, 1950).
Abbrevations
µ : Specific growth rate of yeast (h-1)
µmax : Maximum specific growth rate (h-1)
CiCu : Initial Cu(II) ion concentration (mg/L)
CiNi : Initial Ni(II) ion concentration (mg/L)
Cac,Cu : Bioaccumulated Cu(II) ion concentration at any time
(mg/L)
Cac,Ni : Bioaccumulated Cu(II) ion concentration at any time
(mg/L)
Ks : Saturation constant (g/L)
qCu : Specific Cu(II) uptake defined as bioaccumulated Cu(II)
ion quantity per gram of dried yeast at the end of microbial
growth (mg/g)
qNi : Specific Ni(II) uptake defined as bioaccumulated Ni(II) ion
quantity per gram of dried yeast at the end of microbial
growth (mg/g)
So : Initial sucrose concentration (g/L)
S : Sucrose concentration (g/L)
T : Temperature (°C)
t : Reaction time (h)
X : Dried yeast concentration in feed medium at any time
(g/L)
Xmax : Maximum dried yeast concentration (g/L)
Results and Discussions
Microorganism growth and bioaccumulation
properties were investigated as functions of initial metal
ion and molasses sucrose concentrations at pH: 4.0 and
25°C. The uptake yield (uptake %) was described as the
ratio of bioaccumulated concentration of metal ion at the
end of growth to the initial metal ion concentration. The
results were expressed as the units of bioaccumulated
metal ion concentration (Cac,m: mg/L) and specific metal
ion uptake determined as the amount of metal ion per unit
of dry weight of cells (qm: mg/g), dried cell concentrations
at any time (X: g/L), specific growth rate of yeast (μ: h-1).
The specific growth rate of Candida yeasts was
determined from the slope of ln X versus time plot at the
exponential growth phase. The results indicated that
biomass concentration was related to the metal
concentrations in fermentation medium and the
physiological properties of the yeasts. The ability of metal
uptake by metal adapted yeasts were different due to the
physiological properties of the yeasts. All experiments
were conducted at 25°C. The effect of temperature on
metal bioaccumulation depends on cellular metabolism.
Uptake capacity of heavy metals by microorganisms
decreased at low temperatures whereas high temperatures
could damage cells and reduced uptake levels (Brady &
Duncan 1994).
Effect of initial pH on microbial growth
Effect of initial pH on specific growth rate and
maximum microorganism concentrations of Candida
yeasts was examined in the pH range of 2.0-5.0 at 10 g/L
molasses sucrose concentration. Maximum specific
growth rate and microorganism concentrations were
obtained at pH: 4.0. Initial pH was a major factor in the
quantity of metal ion bioaccumulation. All Candida
species showed growth at pH: 2.0-5.0. The highest value
of specific growth rate and microorganism concentration
was found as 0.308 h-1 and 3.111 g/L respectively using
C. lipolytica in metal free media (Table 1).
Bioaccumulation experiment was conducted at pH: 4.0
which showed maximum growth.
Effect of initial sucrose concentration on microbial
growth
The effect of initial sucrose concentration on growth
rates of Candida yeasts in metal-free media was
investigated in the sucrose concentration range of 1.0-
20.0 g/L, at pH: 4.0 and 25°C. The relationship of specific
growth rate to substrate concentration was explained with
saturation kinetics. It was observed that the specific
growth rate and biomass concentration increased with
increasing initial sucrose concentration up to 20.0 g/L.
Microorganism concentration increased from 1.52 g/L to
3.48 g/L with an increase in the initial sucrose
concentration from 1.0 to 20.0 g/L for C. lipolytica in
metal-free media (Fig. 1). Lower growth performance
among the yeast cells was seen using C. utilis. We have
found that molasses was a suitable carbon source for
fermentation medium of Candida species. It was also
reported in previous studies that molasses could be used
as feasible and economical for microbial growth (Aksu &
Dönmez 2000, Açıkel & Alp 2009, Evirgen & Sağ Açıkel
2014).
70 G. Mersin & Ü. Açıkel
Effect of initial metal ion concentrations on growth of
Candida species
The effect of initial Cu(II) and Ni(II) ion
concentrations on microbial growth of Candida species
were examined at different Cu(II) and Ni(II) ion
concentrations. The metal ion concentrations in the
fermentation the medium varied in the range of 25-250
mg/L for Cu(II) and Ni(II). The range of molasses sucrose
concentrations of prepared fermentation media varied
between 1 and 20 g/L. Both metal ions inhibited specific
growth rates and biomass concentrations for all yeasts.
We found that the inhibition effect of Ni(II) ions on
specific growth rate and microorganism concentration
was higher than Cu(II) ions for all yeasts. Candida
lipolytica showed the highest specific growth rate and
microorganism concentration among the yeasts in metal
media. Maximum specific growth rate significantly
decreased from 0.302 h-1 to 0.278 h-1 with an increase in
the initial Ni(II) concentration from 100 to 200 mg/L for
C. utilis. Inhibition kinetics was determined using the
double reciprocal plot of the Monod equation. When
initial Cu(II) and Ni(II) ion concentrations were increased
in the range of 25-250 mg/L, the maximum specific
growth rate (h-1) of the yeasts decreased whereas
saturation constants (Ks) increased (Table 2).
Table 1. Effect of initial pH on the maximum specific growth rate, maximum dried microorganism concentration in metal-free
medium (So: 10 g/L; T:25°C).
pH
C. membranaefaciens
C. utilis
C. tropicolis
C. lipolytica
µmax (h-1)
Xmax (g/L)
µmax (h-1)
Xmax (g/L)
µmax (h-1)
Xmax (g/L)
µmax (h-1)
Xmax (g/L)
2
0.198
2.001
0.177
1.985
0.206
2.112
0.211
2.223
3
0.241
2.552
0.222
2.443
0.253
2.601
0.261
2.751
4
0.253
2.854
0.238
2.658
0.289
2.999
0.308
3.111
5
0.251
2.851
0.235
2.651
0.279
2.995
0.306
3.005
Fig. 1. Effect of initial sucrose concentration on specific growth rate and microorganism concentration for Candida lipolytica, Candida
utilis, Candida tropicalis and Candida membranaefaciens (pH: 4.0; SR: 150 rpm; T: 25oC).
Table 2. Comparison of the maximum specific growth rates and the saturation constants in the presence of increasing concentrations
of single Cu(II) and Ni(II) ions (So: 1-20 g/L; pH: 4, T: 25°C; SR: 150 rpm.
Metal Ion
C. membranefeciens
C. utilis
C. tropicalis
C. lipolytica
CiCu
(mg/L)
CiNi
(mg/L)
µmax (h-1)
Ks (g/L)
µmax (h-1)
Ks (g/L)
µmax (h-1)
Ks (g/L)
µmax (h-1)
Ks (g/L)
25.0
0.0
0.354
5.399
0.341
5.805
0.374
4.555
0.422
5.080
100.0
0.0
0.337
5.354
0.341
6.472
0.363
4.677
0.388
4.568
200.0
0.0
0.336
6.586
0.299
6.033
0.338
4.887
0.378
5.378
250.0
0.0
0.310
6.771
0.285
6.519
0.313
4.984
0.335
4.920
0.0
25.0
0.346
5.725
0.319
5.600
0.352
4.243
0.345
4.473
0.0
100.0
0.333
5.718
0.302
5.408
0.356
5.013
0.395
5.268
0.0
200.0
0.309
7.004
0.278
6.374
0.309
5.111
0.328
4.918
0.0
250.0
0.267
6.265
0.264
7.196
0.284
5.269
0.317
5.796
Biomass production of Candida species 71
Trakya Univ J Nat Sci, 22(1): 67-76, 2021
Candida utilis was sensitive to high concentrations of
Cu(II) with an extension in lag phase duration, correlated
with a decrease in yeast production. The increase of Cu(II)
and Ni(II) concentrations led to a drastic decrease in
microbial growth for C. utilis. Candida lipolytica was
highly resistant to Cu(II) when compared with three other
yeasts. The increase in Cu(II) concentration also caused a
decrease in biomass production. For instance,
microorganism concentrations were found as 2.854 g/L,
2.658 g/L, 2.999 g/L and 3.111 g/L for C.
membranaefaciens, C. utilis, C. tropicalis and C.
lipolytica, respectively, in the metal-free media (Table 1).
Biomass concentrations decreased as 2.666 g/L, 2.456
g/L, 2.831 g/L and 2.968 g/L for C. membranaefaciens,
C. utilis, C. tropicalis and C. lipolytica, respectively, at
100 mg/L Cu(II) concentration (Table 3). The decrease in
biomass concentration was found higher for fermentation
medium containing 100 mg/L Ni(II). When initial Ni(II)
concentration was 100.0 mg/L, microorganism
concentrations were found as 2.574 g/L, 2.371 g/L, 2.735
g/L and 2.868 g/L for C. membranaefaciens, C. utilis, C.
tropicalis and C. lipolytica, respectively (Table 3).
Bioaccumulation experiments
The combined effect of heavy metals and molasses
sucrose on the bioaccumulation properties of adapted
Candida yeasts was investigated. Initial metal ion
concentration in the fermentation medium varied in the
range 25-250 mg/L for Cu(II) and Ni(II) at changing
sucrose concentrations in the range of 1-20 g/L. We found
that bioaccumulated Cu(II) and Ni(II) ions and microbial
growth increased with increasing initial molasses sucrose
concentrations for all Candida yeasts. The uptake
performance of Cu(II) ions was higher than Ni(II), and C.
lipolytica showed the maximum removal efficiency in a
medium containing both single metal ions. An increase in
sucrose concentration significantly increased the growth
and metal uptake capacity of the microorganisms. The
amount of Cu(II) and Ni(II) ions uptake per gram dry
microorganism increased with increasing the initial metal
concentrations up to 250 mg/L. We found that microbial
growth was supported by increasing initial sucrose
concentrations in metal- stressed fermentation media. In
other words, bioaccumulation of Cu(II) and Ni(II) ions by
Candida species was metabolism dependent. Specific
Cu(II) uptake was determined as 31.77 mg/g, 29.71 mg/g,
33.04 mg/g, and 34.39 mg/g for C. membranaefaciens, C.
utilis, C. tropicalis, C. lipolytica, respectively, at
fermentation media containing 10 g/L constant sucrose
and 200 mg Cu(II) mg/L (Fig. 4). At the same conditions,
the specific Ni(II) uptake significantly decreased for all
yeasts in medium containing Ni(II) ions when compared
to Cu(II) ions. For example, the specific growth rates were
obtained in a medium containing 200 mg Ni(II)/L and 10
g/L sucrose were determined as 25.96 mg/g, 24.76 mg/g,
26.13 mg/g, and 28.80 mg/g for C. membranaefaciens, C.
utilis, C. tropicalis, C. lipolytica, respectively (Fig. 5).
Although removal efficiency increased with increasing
initial sucrose concentration for all yeasts, it decreased
with increasing initial metal ion concentrations. Uptake
efficiency significantly decreased from 88.80% to 56.44%
with an increase in the initial Cu(II) concentration from
25 to 200 mg/L for C. lipolytica at 20 g/L constant sucrose
concentration (Table 4). This decrease may result from
metal-binding sites on yeast surfaces. It was reported that
binding sites were occupied first rapidly, then decreased
with increasing metal concentrations (Honfi et al. 2016).
The bioaccumulated Cu(II) concentrations were
higher than bioaccumulated Ni(II) concentrations in
growth media containing 25 mg/L single Cu(II) and Ni(II)
ions (Figs 2, 3).
The intolerance of living cells to high metal
concentrations limits bioaccumulation process, but living
cells have the potential genetic recombination to improve
the metal-adapted strain (Malik 2004). Heavy metal
bioaccumulation capacity of Candida species was
strongly pH-dependent due to changing solution
chemistry and active functional groups on the biomass.
Due to the Ni(II) inhibition effect, metal uptake
performance decreased from 81.68% to 46.28% with
increasing Ni(II) concentration from 25 to 250 mg/L for
C. lipolytica. According to Gönen and Aksu (2008), C.
utilis accumulated toxic heavy metals. The uptake
efficiency of Cu(II) by C. utlis was observed as 27.0% in
a growth medium containing 11.2 g/L sucrose and 101.3
mg/L Cu(II) ions (Gönen & Aksu 2008). Pawan and Devi
(2018) investigated bioaccumulation of Ni(II), Zn(II),
Cr(VI) by Aspergillus awamori, A. flavus and A. niger and
found that A. niger was a highly tolerant strain. Maximum
dry weight of A. flavus was found as 12.31 g/L for Ni(II)
in medium containing 100 mg/L initial broth
concentration (Pawan & Devi 2018). Rehman and Anjum
(2010) reported that Candida tropicalis was a metal-
resistant yeast. They reported that C. tropicalis
bioaccumulated 64% copper from industrial wastewater
after 4 days (Rehman & Anjum 2010).
Table 3. Comparison of decrease in maximum dried microorganism concentrations obtained in the metal-free fermentation media
with Cu(II) and Ni(II) ions present as the single metal at 100 and 250 mg/L (So: 10 g/L; pH: 4, T: 25°C; SR: 150 rpm).
Metal Ion
C. membranefeciens
C. utilis
C.tropicalis
C. lipolytica
CiCu
(mg/L)
CiNi
(mg/L)
Xmax
(g/L)
Decrease
%
Xmax
(g/L)
Decrease
%
Xmax
(g/L)
Decrease
%
Xmax
(g/L)
Decrease
%
100.0
0.0
2.666
2.4
2.456
7.6
2.831
5.6
2.968
4.6
250.0
0.0
2.335
14.5
2.148
19.2
2.483
17.2
2.607
16.2
0.0
100.0
2.574
5.8
2.371
10.8
2.735
8.8
2.868
7.8
0.0
250.0
2.175
20.4
1.199
54.9
2.315
22.8
2.432
21.8
72 G. Mersin & Ü. Açıkel
Fig. 2. Effect of initial sucrose concentration on the bioaccumulated Cu(II) ion quantity per unit weight of dried biomass and specific
Cu(II) uptake in the presence of 25 mg/L Cu(II) (pH: 4.0; SR: 150 rpm; T: 25°C).
Fig. 3. Effect of initial sucrose concentration on the bioaccumulated Ni(II) ion quantity per unit weight of dried biomass and specific
Ni(II) uptake in the presence of 25 mg/L Ni(II) (pH: 4.0; SR: 150 rpm; T:25°C).
Fig. 4. Effect of initial sucrose concentration on the bioaccumulated Cu(II) ion quantity per unit weight of dried biomass and specific
Cu(II) uptake in the presence of 200 mg/L Cu(II) (pH: 4.0; SR: 150 rpm; T: 25°C).
Biomass production of Candida species 73
Trakya Univ J Nat Sci, 22(1): 67-76, 2021
Fig. 5. Effect of initial sucrose concentration on the bioaccumulated Ni(II) ion quantity per unit weight of dried biomass and specific
Ni(II) uptake in the presence of 200 mg/L Ni(II) (pH: 4.0; SR: 150 rpm; T: 25°C).
Table 4. Comparison of the bioaccumulated Cu(II) and Ni(II), the Cu(II) and Ni(II) metal ion bioaccumulation efficiency in the
fermentation media containing single Cu(II) and Ni(II) ions (pH: 4.0; T: 25°C; SR: 150 rpm.
Yeast
C. membranaefaciens
C. utilis
C. tropicolis
C.lipolytica
Si
(g/L)
Ci,Cu
(mg/L)
Cac,Cu
(mg/L)
Uptake
(%)
Cac,Cu
(mg/L)
Uptake
(%)
Cac,Cu
(mg/L)
Uptake
(%)
Cac,Cu
(mg/L)
Uptake
(%)
1
25
2.50
10.00
2.13
8.52
2.88
11.52
3.13
12.52
10
14.50
58.00
12.60
50.40
16.40
65.60
18.20
72.80
20
17.10
68.40
15.1
60.4
19.67
78.68
22.20
88.80
1
100
6.88
6.88
5.50
5.50
7.56
7.56
8.70
8.70
10
46.80
46.80
38.40
38.40
51.48
51.48
59.20
59.20
20
58.47
58.47
46.77
46.77
64.31
64.31
73.96
73.96
1
200
14.60
7.30
8.65
23.38
16.06
8.03
17.51
8.75
10
79.80
39.90
68.70
20.67
88.20
44.10
96.30
48.15
20
95.88
47.94
83.37
34.35
105.46
52.73
114.96
57.48
1
250
11.60
4.64
8.50
3.40
15.20
6.08
16.42
6.56
10
94.81
37.92
79.01
31.60
104.29
41.71
112.60
45.04
20
115.05
46.02
95.88
38.35
126.56
50.62
141.10
56.44
So
(g/L)
Ci,Ni
(mg/L)
Cac,Ni
(mg/L)
Uptake
(%)
Cac,Ni
(mg/L)
Uptake
(%)
Cac,Ni
(mg/L)
Uptake
(%)
Cac,Ni
(mg/L)
Uptake
(%)
1
25
2.04
8.16
1.51
6.04
2.36
9.44
2.88
11.52
10
12.20
48.80
10.10
40.40
13.50
54.00
16.8
67.20
20
14.71
58.84
12.08
48.32
16.13
64.52
20.42
81.68
1
100
5.84
5.84
4.47
4.47
6.50
6.50
7.39
7.39
10
39.70
39.70
31.20
31.20
44.30
44.30
50.70
50.70
20
49.70
49.70
38.80
38.80
55.31
55.31
62.86
62.86
1
200
10.80
5.40
6.66
3.33
12.05
6.02
13.65
6.82
10
61.20
30.60
53.70
26.85
65.50
32.75
75.80
37.90
20
72.87
36.43
65.50
32.75
79.10
39.55
89.67
44.83
1
250
8.82
3.52
6.63
2.65
10.50
4.20
13.46
5.38
10
72.20
28.88
62.10
24.84
79.26
31.70
92.33
36.93
20
87.44
34.97
78.70
31.48
96.18
38.47
115.70
46.28
The physiological and biochemical properties of
microorganisms can be changed by the presence of heavy
metals. Heavy metals such as copper (Cu(I) and Cu(II))
carry soluble electrons and can catalyze Fenton and
Haber-Weiss reactions. Cytoplasmic molecules in
microorganisms can cause serious damage to DNA, lipids
and other proteins (Giner-Lamia et al. 2014). Heavy metal
can cause ion imbalance by binding to the cell surface and
entering through ion channels or transmembrane carriers
(Chen et al. 2014). High vitamin and mineral contents of
molasses sucrose stimulate microorganisms growth
(Razack et al. 2013). The structure of metal-binding
agents (homopolysaccharides, single saccharides, and
acid components) in microorganisms and their
distributions in the cell wall determine the metal
accumulation capacity (Raspor & Zupan 2006). Heavy
74 G. Mersin & Ü. Açıkel
metal cations can interact with some cations on the cell
wall. The cell wall and heavy metal interactions may
inhibit the function of physiological cations in the cell
structure. As a result of this inhibition, oxidative stress
occurs in the cell. For example, Fe, Zn, and Ca affect the
uptake and toxicity of heavy metals in microorganisms.
Ca and Fe ions reduce the uptake of Cd (Volland et al.
2014). Metal uptake is mainly related to the
concentrations of metals in solution (Modak & Natarajan
1995). Also, intercellular electrostatic interactions in cells
play an important role in metal uptake capacity. At a
certain equilibrium, biomass adsorbs more metal ions at
lower cell concentrations (Gourdon et al. 1990). The
inhibition effect of heavy metals on microorganism
growth depends on the total metal ion concentration, the
chemical structure of the metal, and redox potential of
metal. Environmental factors such as temperature, pH,
organic acids, and humic acids can alter the conversion,
transport, valence of heavy metals, and the resistance of
microorganisms to heavy metals.
Conclusion
In the present study, microbial growth and
bioaccumulation properties of Candida biomasses were
investigated as a function of molasses sucrose and metal
ion concentrations in a batch reactor. Optimum pH was
found 4.0 for each yeast and all experiment conducted at
this pH value. Biomass concentrations and specific
growth rates increased with increasing initial sucrose
concentration in metal and metal-free media. Both Cu(II)
and Ni(II) inhibited specific growth rates and the
contribution of Cu(II) inhibition was lower than Ni(II).
Thus, the maximum specific growth rates of yeasts
decreased with increasing initial metal ion concentrations.
The saturation constants were determined in both metal
and metal-free fermentation medium. Saturation constants
and the amounts of Cu(II) and Ni(II) ions uptake per gram
dry biomass increased with the increase of the initial
concentration of heavy metals ions. The removal
efficiency of Cu(II) ions was higher than Ni(II), and
Candida lipolytica showed maximum uptake efficiency in
fermentation medium containing Cu(II) and Ni(II) ions.
Results showed that bioaccumulation of Cu(II) and Ni(II)
ions by Candida biomass was metabolism dependent.
Candida biomasses showed different bioaccumulative
capacities for the same metal ions. The yeast surface
properties and cell wall components may change affinity
and cell-metal interaction. In addition, environmental
conditions can affect the accumulation capacity of yeasts.
Although Cu(II) and Ni(II) have similar chemical
properties, genetic differences of yeasts and the chemical
nature of metals caused differences in bioaccumulation
performances. This study showed that Candida biomasses
are useful for removal Cu(II) and Ni(II) ions from aqueous
solutions. The comparison of bioaccumulation properties
of Candida yeasts may help to an effective selection of
Candida strains for water treatments.
Ethics Committee Approval: Since the article does
not contain any studies with human or animal subject, its
approval to the ethics committee was not required.
Author Contributions: Execution: G.M., Data
analysis/interpretation: Ü.A., Writing: G.M., Critical
Review: Ü.A.
Conflict of Interest: The authors have no conflicts of
interest to declare.
Funding: This research was supported by Cumhuriyet
University Scientific Research Projects Unit (Project
Number: BAP- M-354).
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