Environmental Technology & Innovation 20 (2020) 101057

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Environmental Technology & Innovation

journal homepage: www.elsevier.com/locate/eti

Bioaccumulation of Ni(II) on growing cells of Bacillus sp.:

Response surface modeling and mechanistic insight
∗ ∗∗
Animesh Naskar a,b , , Rajib Majumder c , , Mitrabrata Goswami b
a
Department of Food Technology and Biochemical Engineering, Jadavpur University, Kolkata 700 032, India
b
Department of Food Technology, Hemnalini Memorial College of Engineering, MAKAUT, Kalyani 741246, India
c
Department of Biotechnology, School of Life Science and Biotechnology, Adamas University, Kolkata 700126, India

article info a b s t r a c t

Article history: The growing cells of Bacillus cereus M116 , depending on the growth phase of culture was
Received 11 January 2020 found to bioaccumulate Ni(II) from its aqueous solution to the extent of 80% wherein
Received in revised form 12 July 2020
surface binding (∼60%) dominated over intracellular accumulation (∼20%). No growth
Accepted 13 July 2020
Available online 18 July 2020
was observed in the metal ion concentration beyond 50 mg L−1 . The hydroxyl, amine,
carboxylate and phosphate groups of cell surface were the most important moieties in
Keywords: chemical interactions with Ni(II) ions as measured by FTIR. The surface characteristics
Bacillus cereus M116 of cells were investigated meticulously by FE-SEM equipped with EDXA indicating
Nickel conspicuous changes due to metal accumulation. The potential effects of the process
Bioaccumulation variables [initial pH (A), temperature (B), inoculum volume (C) and medium volume (D)]
Response surface methodology
were optimized using Response Surface Methodology (RSM) followed by Box–Behnken
FE-SEM
design to study the bioaccumulation phenomenon. The maximum Ni(II) accumulation
FTIR
was noted at 6.5, 32.5 ◦ C, 2.5% and 50 mL corresponding to pH, temperature, inoculum
volume and medium volume, respectively. The most significant factors including one
independent variable (D) and two quadratic effects (B2 and D2 ) were identified by
ANOVA. The experimental response under these optimum conditions was found to be
78.76% which closely matched the yield (78.52%) predicted by the statistical model with
R2 = 0.8790.
© 2020 Elsevier B.V. All rights reserved.

1. Introduction

Pollution of water bodies with heavy metals, which represents a serious environmental problem, is escalating at
an alarming rate due to increase in industrial activities (Naskar et al., 2020). The divalent nickel cation is an essential
micronutrient, but poses severe toxicity to human health at higher concentrations (Khadim et al., 2019; Khan et al.,
2019; Naskar et al., 2016). Being non-biodegradable, this metal accumulates into living cells through the ecological
food chain and acts as a mutagen, carcinogen, and embrayotoxin (Fawzy et al., 2016). Various industries such as
battery, paint formulation, electroplating, etc. routinely discharge an alarming amount of this metal into the environment
through wastewater (Dotto et al., 2016). The maximum permissible limit of divalent nickel is 3 mg L−1 in effluent before
disposal into natural waters as directed by Environmental Regulatory Authority, India (Naskar and Bera, 2018). So as to
eliminate the metals from aqueous solutions early conventional attempts, such as chemical precipitation, ultrafiltration,

∗ Corresponding author at: Department of Food Technology, Hemnalini Memorial College of Engineering, MAKAUT, Kalyani 741246, India.
∗∗ Corresponding author at: Department of Biotechnology, School of Life Science and Biotechnology, Adamas University, Kolkata 700126, India.
E-mail addresses: animesh.ftbe@gmail.com (A. Naskar), rajib.iicb@gmail.com (R. Majumder).

https://doi.org/10.1016/j.eti.2020.101057
2352-1864/© 2020 Elsevier B.V. All rights reserved.
2 A. Naskar, R. Majumder and M. Goswami / Environmental Technology & Innovation 20 (2020) 101057

reverse osmosis or ion exchange, can be employed either individually or together. However, these methods have several
constraints for widespread applications (Beni and Esmaeili, 2019; Cao et al., 2018; Khajavian et al., 2019). This has led to
the adoption of biosorption and/or bioaccumulation as an effective alternative in terms of performance, sludge production,
cost, and most desirably eco-friendly nature (Hasan et al., 2016; Li et al., 2019, 2017).
In this context, as microbial cells have a large surface area and many active sites, they are able to effectively reduce the
mobility and lock up target metals (Ganguly et al., 2011). Direct utilization of growing cells permits a simpler framework
for control along with lower operational expenses (Chojnacka, 2010; Huang et al., 2014). Hence, the application of growing
cells may pave the way for the Ni(II) bioremediation from wastewaters. Only a few reports are available on bacterial
systems using growing cultures based on our literature survey (Ganguly et al., 2011; Malik, 2004). For the first time, we
report the accumulation behavior of Ni(II) on growing cells of mutated Bacillus sp. The process can be ineffective under
extreme physicochemical conditions such as high salt concentrations, extreme pH of the medium and large amounts of
heavy metals in the system (Malik, 2004). However, the growing cells allow initial rapid extracellular metal attachment
followed by slower metabolic dependent intracellular accumulation of metal ions (El-Helow et al., 2000; Srinath et al.,
2002; Yilmaz and Ensari, 2005).
For any bioprocess to be implemented, a reliable statistical approach is required to advance the basic experimental
conditions. The response surface methodology (RSM) based on the polynomial equations offers mathematical and
statistical approaches that have been successfully employed in this present study. The RSM strategy has also proven its
worth in terms of time, the number of experiments, economic viewpoint, accuracy and contemplates interactions between
parameters on the response (Majumder et al., 2017; Shi et al., 2015). Thus, the optimum state of each parameter is to be
explored to achieve a better performance of the system.
The present study focused on the accumulation characteristics of toxic Ni(II) ions by experimentally selected growing
cells of Bacillus cereus M116 . The considerable attention was directed to RSM study to optimize as well as an in-depth
understanding of the importance of different process parameters. Besides, it was intended to explore the mechanisms of
Ni(II) accumulation with growing bacterial cells through field emission scanning electron microscope (FE-SEM) equipped
with energy dispersive X-ray analyzer (EDXA) and Fourier transform infrared (FTIR) spectrophotometer.

2. Materials and methods

2.1. Chemicals/reagents

All the chemicals/reagents of analytical grade were purchased from E-Merck, India. Microbiological ingredients/media
used in this experiment were procured from Himedia, India.

2.2. Microorganisms

The strains of Escherichia coli, Pseudomonas aeruginosa and Bacillus cereus used in this experiment were obtained from
Microbial Type Culture Centre, Institute of Microbial Technology, Chandigarh, India. This particular strain Bacillus cereus
M116 was isolated, mutated in the laboratory and patented in IMTECH, India (Ganguly et al., 2011).

2.3. Screening of microorganisms

During initial screening, experiments were carried out in 250 mL Erlenmeyer flasks containing sterile growth medium
[0.1% beef extract, 0.2% yeast extract, 0.5% NaCl, and 0.5% peptone in (w/v)] (pH 6.0 and 6.5 for each strain). The media
containing stipulated concentrations of Ni(II) (10 mg L−1 ) were inoculated with 2% inoculums (w/v) of different bacterial
strains separately and incubated at 30 o C for 72 h under shaking condition (120 rpm). At the end of incubation, each
mixture was centrifuged (5500 rpm for 10 min) to separate the cells and residual concentration of Ni(II) was measured
by atomic absorption spectrophotometer (Chemito Technologies Pvt. Ltd, India, Model no. AA 203, wavelength: 232 nm,
slit width: 0.5 nm). The following equation (Eq. (1)) was used to find out the efficiency (%) of metal removal of each strain.

C0 − Ce
Remov al(%) = × 100 (1)
C0
where C0 and Ce represent the initial and equilibrium metal ion concentration, respectively.

2.4. Minimum inhibitory concentration and bacterial growth measurement

To evaluate the minimum inhibitory concentration of Ni(II) ions, bacterial cells (Bacillus cereus M116 ) were grown in
presence of different Ni(II) concentrations (10–100 mg L−1 ) at 30 o C for 72 h under shaking condition (120 rpm). For
each study, the control experiment without Ni(II) in the growth medium was also maintained. During the experiments,
the microbial growth was analyzed by measuring optical density of solution (OD at 600 nm) at regular time intervals
using a UV–Vis spectrophotometer (Perkin-Elmer, Singapore) and growth curve was also determined. Each data point
was collected from an individual flask and therefore no correction was necessary for the withdrawal of sampling volume.
The experiments were executed in triplicates, and the mean value is depicted in the section of results and discussion
(Akhtar et al., 2018; Huang et al., 2018; Kuippers et al., 2018).
 A. Naskar, R. Majumder and M. Goswami / Environmental Technology & Innovation 20 (2020) 101057 3

Table 1
Experimental ranges and levels in the RSM design.
Factors Range and level
−1 0 +1
A: pH 5.5 6.5 7.5
B: Temperature (◦ C) 25 32.5 40
C: Inoculums volume (%) 1 2.5 4
D: Medium volume (mL) 25 50 75

2.5. Batch bioaccumulation experiments using Response Surface Methodology based on Box–Behnken design

The Box–Behnken design for four independent variables [pH (A), temperature (B), inoculum volume (C) and medium
volume (D)], each at four levels, was used to develop a second-order polynomial model for determining the optimum
levels. A total of twenty-nine experimental runs with various combinations of the variables were conducted to obtain the
desired response [Ni(II) removal percentage]. The highest and lowest limits of the independent variables were shown in
Table 1. The initial concentration of Ni(II) in the medium was adjusted to 30 mg L−1 . In each case, the differences in Ni(II)
concentration before and after bioaccumulation were used to find out the efficiency (%) of metal removal by the growing
cells of Bacillus cereus M116 as Eq. (1). The behavior of each variable, their interactions, and statistical analysis to attain
predicted responses were explained by the following second-order polynomial equation (Eq. (2)):
∑ ∑ ∑
Y = βo + β i xi + βij xi xj + βii x2i (2)

where Y is the predicted response, βo is offset term, βi isa linear effect, βii is squared effect, βij is an interaction effect,
and xi is the ith independent variable. xi xj and x2i symbolize the interaction and quadratic terms, respectively (Hu et al.,
2016; Majumder et al., 2015, 2017; Noormohamadi et al., 2019; Podder and Majumder, 2016).
All the experimental data were analyzed meticulously using Design-Expert software (Version 10.0, stat-Ease, Inc.,
Minneapolis, United States). The fittest and the accuracy of the model were evaluated by F value and the regression
coefficient (R2 ). The optimal condition for percent Ni(II) removal by the growing cells of Bacillus cereus M116 was determined
by solving the regression equations and the 3D responses for the different parameters using the software.

2.6. Intracellular localization of bioaccumulated nickel

Ni(II) laden cells were harvested by centrifugation and washed twice with milli-Q water. Each experiment was carried
out in a pair of two samples: (i) one sample of metal loaded cells was treated with chelating agent (0.1 M EDTA) for 30
min at 30 ◦ C under shaking condition (180 rpm). Afterward, the cells were separated by centrifugation (5500 rpm for 10
min) and the supernatant containing Ni(II) was determined by atomic absorption spectrophotometer (AAS) to obtain the
amount of Ni(II) ions accumulated on the bacterial surface. Intracellular uptake of Ni(II) ions was measured by digested
the separated pellets with trace-metal grade nitric acid (70%) at 30 ◦ C overnight. The separated pellets were also dried
before acid digestion. (ii) Another sample of the metal loaded cells was dried and followed by direct acid digestion to
assess the amount of Ni(II) accumulation by the whole cells.

2.7. Determination of cell surface functional groups involved in Ni(II) removal by Fourier Transform Infrared (FTIR) spectroscopy
study

To determine the surface functional groups of selected bacterial strains, the FTIR study was conducted (Majumder
et al., 2017; Naskar et al., 2016; Naskar and Majumder, 2017). In brief, the cells were grown with and without Ni(II)
ions in the medium. Afterward, cells were harvested by centrifugation and dried through lyophilization. Both pristine
and metal loaded biomass was powdered and examined using KBr disk technique. The data was recorded in the range of
4000–400 cm−1 with the resolution of 4 cm−1 using Shimadzu IR-Prestige-21 (Japan) spectrophotometer.

2.8. Surface chemistry analysis by FESEM-EDX

Field emission scanning electron microscope (FE-SEM) (Model: JSM-6700F, JEOL, Japan) was employed to explore the
surface topography/ultrastructure of both pristine and metal treated bacterial cell. The presence of various elements was
quantified through EDXA profiling (Majumder et al., 2017; Wu et al., 2015).
4 A. Naskar, R. Majumder and M. Goswami / Environmental Technology & Innovation 20 (2020) 101057

Fig. 1. Bioaccumulation phenomenon of Ni(II) by growing cells of different microorganisms incubated at 30 o C with an initial metal concentration
of 10 mg L−1 : ±SD shown by the error bar.

3. Results and discussion

3.1. Screening of bacterial cells

As mentioned earlier, the growing cells of various microorganisms like Escherichia coli, Bacillus cereus, Pseudomonas
aeruginosa, and Bacillus cereus M116 were taken for initial screening to evaluate the efficacy of Ni(II) bioaccumulation.
Among them, Bacillus cereus M116 claimed its suitability displaying ∼94% of accumulation efficacy at pH 6.5 (Fig. 1), thereby
selected for further experiments. Other cells exhibited the percentage accumulation ranging from ∼74% to ∼89% within
pH 6–6.5. The difference in Ni(II) accumulation capacities may be due to the nature of active accumulation sites of the
living cells and their morphological characteristics (El-Helow et al., 2000; Podder and Majumder, 2016).

3.2. Effect of the growth phase and toxicity of Ni(II)

Heavy metals at high concentrations are considered to be toxic for living microorganisms by damaging the cell
membranes and structure of DNA, disrupting cellular functions and altering enzyme specificity (Açıkel and Alp, 2009).
Hence, its effect on the growth as well as accumulation capacity of selected bacterial strain was investigated and the
respective results are depicted in Fig. 2. This is also established that Bacillus cereus M116 cells at the exponential phase
showed the highest bioaccumulation of Ni(II) ions from its aqueous phase. El-Helow et al. reported maximum uptake
of cadmium ions on Bacillus thuringiensis at the logarithmic phase (El-Helow et al., 2000). On the other hand, cadmium
accumulation by Citrobacter sp. at mid-logarithmic phase was also reported by Macaskie and Dean (1984). According
to the result depicted in Fig. 2(a), an increase in Ni (II) ions concentration from 10 to 20 mg L−1 , the bioaccumulation
capacity of concern growing cells decreases from ∼94% to ∼82%, respectively. There is almost 80% of metal accumulation
occurred at the concentration of 30 mg L−1 of metal ions in the system. Further increase in metal concentration, noticeable
declination in accumulation capacity was found to be observed and no growth of microbial cells was noted in the metal ion
concentration beyond 50 mg L−1 . Therefore, 30 mg L−1 of Ni(II) ion was selected for subsequent experiments. Additionally
increasing metal ions concentration in the growth medium declined the absorbance relating to microbial growth as well
as the metal bioaccumulation (Srinath et al., 2002) as illustrated in Fig. 2(b).

3.3. Metal bioaccumulation

The bioaccumulation process consists of both passive and metabolically dependent active strategies, which can be
considered for growing cells to remove the metal ions from aqueous solution. Usually, sorption is a passive phenomenon,
which characteristically indicates more complex metal binding mechanisms. The sorbate–sorbent interactions virtually
depend on the physicochemical nature of cell wall constituents. According to the composition of growth medium relevant
genetic elements are being expressed to polypeptides for the phenotypic architecture of cell walls (Srinath et al., 2002).
In contrast, the metabolically active process of metal bioaccumulation can be defined as slower dynamic phenomena
including intracellular sequestration as well as detoxifying essential enzymes (Goel et al., 2009). However, the rapid
 A. Naskar, R. Majumder and M. Goswami / Environmental Technology & Innovation 20 (2020) 101057 5

Fig. 2. Effect of initial metal ion concentration on Ni(II) removal (a) and growth curve of Bacillus cereus M116 in presence of respective metal ion
concentrations (b).

Fig. 3. Localization (cell surface and intracellular) of bioaccumulated Ni(II) in Bacillus cereus M116 .

sorption of metal ions by cell surface should be much higher in quantity than intracellular accumulation by growing
cells as expected. The experimental findings were more consistent with such behavior showing metal ions were removed
using Bacillus cereus M116 by ∼60% involving cell surface active sites, while ∼20% Ni(II) ions was accumulated internally
(Fig. 3). Hence, it may be articulated that the surface sorption dominate over internal accumulation of Ni(II) ions by the
selected bacterial strain.

3.4. Biomass characterization

3.4.1. FTIR study revealed the role of major functional groups in Ni(II) removal
Pristine bacterial cells demonstrate the distinct peaks corresponding to their relevant functional groups, represented
by the blue line in Fig. S1. Upon treated with Ni(II) ions, several peak positions were changed appreciably to a higher
or lower frequency as denoted by the red line. Also, major functional groups involved in metal binding are shown
with the green arrows (Fig. S1). Peak positioned at 3405.86 cm−1 and 3269.12 cm−1 which are characteristic peaks of
bonded –OH of polysaccharides and –NH stretching vibration of amine groups, have been shifted to 3439.44 cm−1 and
3291.51 cm−1 , respectively (Hasan et al., 2016). Further, the signals observed at 1453.02 cm−1 (O–H deformation in the
carboxyl groups or N–H bending of amine group), 1400.18 cm−1 [symmetric stretching vibration of deprotonated carboxyl
groups (COO− )], and 1229.07 cm−1 (stretching vibration of P=O of C–PO23− groups) have been shifted respectively to
1443.94 cm−1 ,1413.59 cm−1 and 1242.48 cm−1 . Interestingly, the transmittance at 2835.71 cm−1 positions (C–H stretching
vibration of –CH2 and –CH3 groups) in the pristine cell was not found in Ni(II) treated cell (Fawzy et al., 2016). Thus,
FTIR study indicates that the hydroxyl, amino, carboxylate and phosphate groups expressing on the growing cells of
Bacillus cereus M1 16 played a major role for effective removal of Ni(II) ions from its aqueous solution through electrostatic
6 A. Naskar, R. Majumder and M. Goswami / Environmental Technology & Innovation 20 (2020) 101057

Table 2
ANOVA analysis for response surface second order model in relation to Ni(II) bioaccumulation.
Source ANOVA for response surface quadratic model
Sum of df Mean F p-value Remark
squares square value Prob> F
Model 1419.51 14 101.39 3.74 0.0095 Significant
A—pH 45.24 1 45.24 1.67 0.2174
B—Temperature 9.075E−003 1 9.075E−003 3.346E−004 0.9857
C—Inoculum Volume 65.57 1 65.57 2.42 0.1423
D—Medium Volume 177.10 1 177.10 6.53 0.0229 Significant
AB 10.37 1 10.37 0.38 0.5463
AC 6.71 1 6.71 0.25 0.6267
AD 122.10 1 122.10 4.50 0.0522
BC 19.62 1 19.62 0.72 0.4093
BD 62.17 1 62.17 2.29 0.1523
CD 27.93 1 27.93 1.03 0.3274
A2 69.53 1 69.53 2.56 0.1317
B2 416.98 1 416.98 15.37 0.0015 Significant
C2 38.23 1 38.23 1.41 0.2549
D2 624.43 1 624.43 23.02 0.0003 Significant
Residual 379.69 14 27.12
Lack of Fit 379.61 10 37.96 1755.82 <0.0001 Significant
Pure Error 0.086 4 0.022
Core Total 1799.21 28

Table 3
Statistical analysis for the removal of bivalent nickel by RSM.
Std. Dev. 5.21 R2 0.8790
Mean 68.78 Adj R2 0.7700
C.V. % 7.57 Pred R2 −0.2154
PRESS 2186.67 Adeq Precision 6.846

interactions complexation and/or chelation (Hosomomi et al., 2016; Naskar and Bera, 2018; Naskar et al., 2016; Podder
and Majumder, 2016).

3.4.2. FE-SEM and EDX analyses

FE-SEM micrographs of the pristine bacterial cell and Ni(II) laden cells are presented in Fig. S2. It is exhibited that the
topography of the bacterial cell surface has been changed appreciably after metal accumulation [Fig. S2(c)]. Upon exposure
to Ni(II) ions, cells appeared bulge with an uneven surface. This alteration in surface morphology is due to an obvious
response to metal toxicity. Bioaccumulation of Ni(II) ions onto the cell surface was further confirmed by EDX analysis. Fig.
S2(b) exhibited the characteristic EDX pattern for a pristine cell as a result of X-ray emissions based on the high-energy
electron beam. The occurrence of the typical signals of Ni(II) is noted in the EDX spectra for bioaccumulation of metal ions
[Fig. S2(d)] and the findings correlated to the elemental composition reveal the complete disappearance of magnesium
and calcium with concomitant appearance of nickel peak. This can be assumed that the ion-exchange mechanism was
involved for effective metal bioaccumulation by growing cells of Bacillus cereus M116 .

3.5. Process optimization by statistical approach

3.5.1. Box–Behnken analysis

The model predicted responses have been correlated substantially to the actual response in terms of percent removal
of Ni(II) (Table S1). The variance analysis for Ni(II) removal of the BBD (Box–Behnken Design) model has been tabulated
in Table 2. The relationship between response (bioaccumulation) and variables is as follows:
Bioaccumulation (Y) = +78.52+1.94A-0.028B+2.34C+3.84D-1.61AB-1.29AC+5.53AD-2.21BC+3.94BD-2.64CD-3.27A2
-8.02B2 -2.43C2 -9.81D2
The Model F-value of 3.74 suggests that the model is significant. The model terms are also to be significant if the value
offered by ‘‘Prob> F’’ is less than 0.050. In this case, one independent variable (D) and two quadratic factors (B2 and D2 )
were found to be significant model terms as these have a direct influence on the responses (Table 3). Further, the signal
to noise ratio can be measured using ‘adequate precision’. According to statistical analysis, the ratio was established to
6.846, referring that this model can be employed to navigate the design space. Alongside, the regression coefficient (R2 )
and adjusted-regression coefficient (adj-R2 ) were found to 0.8790 and 0.77, respectively (Table 3). The phenomenon can
be exemplified by the fact that the predicted responses are in close agreement with the actual responses obtained from
twenty-nine sets of experiments regarding Ni(II) bioaccumulation by growing cells of Bacillus cereus M116 . This relationship
 A. Naskar, R. Majumder and M. Goswami / Environmental Technology & Innovation 20 (2020) 101057 7

Fig. 4. The 3D response surface plot exhibiting the effect of process variables for Ni(II) bioaccumulation.

of the regression model is also depicted in the ‘‘actual vs. predicted plot [Fig. 5(a)]. Besides, the simultaneous effects of
four parameters on Ni(II) removal are represented by the 3D surface plots (Fig. 4).
The optimized values of the process variables [(A) pH =6.5, (B) temperature =32.5 ◦ C, (C) inoculum volume=2.5% and
(D) medium volume=50 mL] were located within the experimental range culminating to maximum Ni(II) bioaccumulation.
In the present statistical model, the experimental response was 78.76% (under these optimum conditions) which closely
matched the predicted yield (78.52%). Finally, we also validated the RSM derived data with five replicates which resulted
in average Ni(II) bioaccumulation of 77.3 ± 0.87%.

3.5.2. Analysis of physicochemical process variables through perturbation plot

Fig. 5(b) of the response design depicts the perturbation plot for each factor moved or changed from the reference
point. From this plot, it can be determined whether the factors influenced on the Ni(II) bioaccumulation process. The
solution pH (A) not only changes the surface characteristics of growing cells and also affect metal chemistry. In the
8 A. Naskar, R. Majumder and M. Goswami / Environmental Technology & Innovation 20 (2020) 101057

Fig. 5. Predicted vs. actual response plot and (a), perturbation plot of four model parameters obtained from Box–Behnken analysis (b).

previous experiments, it was found that the surface sorption of Ni(II) ions on growing cells dominated over intracellular
accumulation (Fig. 3). For that reason, the impact of various functional groups on the cell surface with the variation of
pH is crucially important for Ni(II) removal. At low pH, (pH—5.5), the metal accumulation was noted to merely 72% due
to exposure of both protonated and deprotonated species corresponding to dissociation of existing functional groups
on the cell surface (Fig. 5b). Deprotonated moiety helps in the binding of Ni(II) ions while protonated groups exhibit
the opposite phenomenon in the medium. As an increasing pH from 5.5 to 6.5, enhanced surface negativity facilitates
more electrostatic interaction with Ni(II) ions causing the better accumulation. Further increasing the pH, the decreasing
trend of metal removal was observed. This behavior may be due to the formation of metal hydroxide in the solution. The
obtained results are more consistent with earlier reports (Deng et al., 2003; Vijayaraghavan and Yun, 2008). The effect
of temperature on Ni(II) accumulation by growing bacterial cells is shown to have pretentious since it is associated with
biological processes regarding microbial growth and metabolism (Fan et al., 2014). Our result demonstrates that the Ni(II)
accumulation efficiency increased initially from 64.97% to 78.52% with the increase in temperature from 25 o C to 32.5 o C,
respectively. Afterward, deterioration in metal removal was found to 69.89% at increasing temperature to 40 o C. This is
probably due to damage to the physical structure of the cells (Park et al., 2010). To ensure whether inoculum volume
had a crucial role in the accumulation phenomenon, the experimental result was also analyzed by the perturbation plot
(Fig. 5b). According to the result, the accumulation of Ni(II) ion increases with an increase in inoculum volume from 1%
and attain optimum at 2.5%. This fact may be linked to the rising of biomass quantity in the growth medium, which has
a positive impact on the Ni(II) ion accumulation. Further increment of inoculum volume (4%) in the medium governs
a negative influence on metal removal. The obtained decreasing behavior in later stages may be due to the limiting of
nutrients in the growth medium (Ganguly et al., 2011). Another important process variable, medium volume displays its
significant effect on metal accumulation according to the perturbation plot. Results show that the accumulation of Ni(II)
ions increases with an increase in medium volume from 25 mL to 50 mL. A fewer cell formation in the growth medium
is the most possible reason for the low percentage of metal removal using 25 mL of medium. Interestingly, high medium
volume (75 mL) also decreases the metal accumulation probably due to diminishing the aeration rate in the system,
causing the inhibition of the microbial growth (Ganguly et al., 2011).

4. Conclusions

In conclusion, the growing cells of Bacillus cereus M116 were found to have substantial Ni(II) bioaccumulation efficiency
from its aqueous solution where surface binding of metal was much higher than the internal accumulation. The
involvement of hydroxyl, amino, carboxylate and phosphate groups on the cell surface were identified for binding the
Ni(II) ions through the FTIR study. Further, FESEMI–EDXA analyses confirmed the incorporation of Ni(II) ions into the
cells surface. Optimum physicochemical parameters obtained from response surface methodology (RSM) demonstrated
that the values obtained from experiments were agreed well to the predicted responses with the correlation coefficient of
0.8790. According to the statistical analysis report, the independent model term D (medium volume) and two quadratic
effects (B2 and D2 ) of RSM design were significant with the R-square (R2 ) value of the regression model is 0.8790.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
 A. Naskar, R. Majumder and M. Goswami / Environmental Technology & Innovation 20 (2020) 101057 9

Acknowledgments

Author, Animesh Naskar gratefully acknowledges the University Grants Commission (UGC), India for providing the
fund to accomplish the research work. Honorable Chancellor of Adamas University is duly acknowledged for providing
infrastructure facility to Rajib Majumder.

Appendix A. Supplementary data

Supplementary material related to this article can be found online at https://doi.org/10.1016/j.eti.2020.101057.

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