Degradation of Cefdinir by Candida Sp.
SMN04
and MgO Nanoparticles—An Integrated (Nano-Bio)
Approach
Adikesavan Selvi and Nilanjana Das
School of Bio Sciences and Technology, VIT University, Vellore 632014, Tamilnadu, India; nilanjana00@lycos.com (for
correspondence)
Published online 27 November 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/ep.12279
The present study evaluates the effect of integrated nano-
bio approach involving nanoscale magnesium oxide (n-
MgO) and yeast Candida sp. SMN04 on cefdinir degradation
in aqueous medium. The nanoparticle was chemically syn-
thesized and characterized by atomic force microscopy
(AFM), scanning electron microscopy (SEM), EDAX analysis,
and particle size analyser. Nano-bio integrated system was
prepared using chemically synthesized n-MgO (10 mg
mL21), coated onto the surface of yeast cells without causing
any lethal effects to the cell. Cefdinir (250 mg L21) degrada-
tion was studied using individual and nano-bio integrated
system both. Nano-bio integrated system was found to be
more effective for cefdinir degradation compared to native
yeast cell. The adherence of nanoparticles on the surface of
the yeast cells increased the permeability of the cell mem-
brane, thereby enhancing the entry of cefdinir into the cell.
The kinetic data showed the half-life of cefdinir, which was
1.46 days for integrated system and 2.97 days for native
yeast, which confirmed that the integrated approach reduced
the half-life to less than half of the time taken by the yeast
alone. This study signifies the potential efficacy of the nano-
bio integrated approach to serve as an effective remedial tool
for the treatment of pharmaceutical wastewater. V C 2015 Ameri-
can Institute of Chemical Engineers Environ Prog, 35: 706–714, 2016
Keywords: cefdinir, degradation, magnesiumoxide nano-
particles (n-MgO), nano-bio integrated system, yeast
INTRODUCTION
In recent years, it has become clear that pharmaceuticals
are an important group of environmental pollutants [1]. Phar-
maceutical industries involved in the production of antibiot-
ics discharge their wastes openly, which contains some
quantity of these active compounds that are toxic in nature.
The accumulation and persistence of antibiotics in the envi-
ronment produce harmful effects, even at low concentration
in which they are detected [2].
Cefdinir is an advanced third generation semi-synthetic
cephalosporin antibiotic, characterized by a vinyl group at C-
3 and a (Z)-2-(2-amino-4 thiazolyl)-2-(hydroxyimino) acetyl
moiety at C-7 and used for the treatment of acute respiratory
related disorders and mild skin infections. The effluents
released from cephalosporin production units are reported to
C 2015 American Institute of Chemical Engineers
V involves the combined treatment of biological system with
706 May 2016 Environmental Progress & Sustainable Energy (Vol.35, No.3) DOI 10.1002/ep
release harmful compounds which are resistant to degrada-
tion, photo-transformation, and natural degradation [3]. The
presence of high concentration of cephalosporin in the envi-
ronment leads to very high chemical oxygen demand, thus
by increasing the toxic strength of the effluent [4].
Contamination of surface and ground waters by synthetic
antibiotic compounds is considered as a potential threat to
human and ecological health [5]. Over the past 2 decades,
there have been an increasing number of infections world-
wide due to third-generation cephalosporin-resistant (3GCs-
R) Escherichia coli isolates [6–8].
In the last few decades, considerable attention has been
given to the treatment of pharmaceutical wastewater. A wide
range of physico-chemical methods are being used for the
treatment of pharmaceutical wastewaters which are of lim-
ited applicability because of the limitations such as ineffi-
ciency of remediating high strength wastewater, high
operating cost, huge labor requirement, high equipment
cost, intervention of toxic by-products, etc. [2]. Bioremedia-
tion using yeasts has attracted special interest in the present
study since yeast species are found to be adaptable to chang-
ing environmental conditions, persist in natural habitats and
polluted sites, degrade various toxic and stable organic sub-
stances like, pharmaceutical compounds etc. [9,10].
Biological treatments always have more advantages com-
pared to other physico-chemical treatments. Biodegradation
of cephalosporin antibiotics using bacteria have been
reported by few researchers [11,12]. The efficiency of yeasts
viz., Peudozyma sp. SMN01, Ustilago sp. SMN02, Ustilago sp.
SMN03, and Candida sp. SMN04 toward cefdinir biodegrada-
tion have been reported in our previous studies [9,13,14].
In the past few years, MgO nanoparticles have attracted
the interests of researchers for its remarkable properties and
important industrial uses such as industrial waste water
remediation in the removal of pharmaceuticals, toxic waste,
toxic gas, paints, semiconductors, and heavy metals [15–17].
Magnesium oxide (MgO) is a heterogeneous catalyst that has
good catalytic potential for degradation reactions of organic
pollutants [18–20]. MgO nanoparticles can act by adsorption
process or direct cell membrane penetration to facilitate the
removal of drug resistant microbes from the polluted envi-
ronment [20,21].
In recent years, few researchers proposed a new approach
toward degradation of environmental contaminants, which
nanoparticles [19,22]. Integrated system may serve as an effec-
tive remediation tool for the treatment of pharmaceutical
wastewater containing cephalosporin antibiotics. This is the
first report on using integrated nano-bio system composed of
n-MgO coupled with a cefdinir degrading yeast Candida sp.
SMN04 for a faster degradation of cefdinir from aqueous
environment.
MATERIALS AND METHODS
Chemicals
Cefdinir (99% purity) was kindly donated by Orchid Phar-
maceuticals, Chennai, India. Dimethyl sulfoxide (DMSO) pro-
cured from SRL Chemicals, India Ltd., was used to prepare a
stock solution of cefdinir (10 g L21). Toluene, magnesium
chloride, and potassium were of analytical grade and are
obtained from Hi-Media India Ltd., SRL Chemicals India Ltd.
and Sigma-Aldrich Chemicals Co. (USA).
Microorganism and Culture
The cefdinir degrading yeast, Candida sp. SMN04
(KF963314.1) isolated from pharmaceutical wastewater [9]
was used in this work. The yeast was maintained on yeast
extract peptone dextrose (YEPD) slants supplemented with
100 mg L21 cefdinir. The degradation studies of cefdinir
were carried out in mineral broth (MB) containing the fol-
lowing per litre: ammonium sulfate 5 g, potassium dihydro-
gen phosphate 1 g, dipotassium hydrogen phosphate 2 g,
magnesium sulfate 0.5 g, sodium chloride 0.1 g, manganese
chloride 0.01 g, ferrous sulfate 0.01 g, sodium molybdate
0.01 g, pH 7.0 6 0.2.
Synthesis of MgO Nanoparticles
Synthesis of MgO nanoparticles (n-MgO) was carried out via
non aqueous sol–gel process following the method by Athar
et al. [22] . Alkali solution of toluene was prepared by dissolving
2 g of KOH (35.71 mmol) pellets in 20 mL of toluene with vigor-
ous stirring for 12 h, followed by the addition of 3 g (14.75
mmol) of MgCl2 6H2O into this solution and then refluxed for
3 h. The compound was separated out by filtration, and then
washed thoroughly with distilled water, until the filtrate became
neutral. The compound was oven dried for 6 h at 1008C fol-
lowed by calcination at 5508C (with an increase in temperature
of 28C per min for 4 h) in atmospheric condition. The white fine
powder with 85% yield was obtained.
MgCl2 : 6H2 O 1 2KOH ! MgO 1 2 KCl 1 7 H2 O (1)
Characterization of n-MgO
X-Ray Diffraction
The X-ray diffraction (XRD) patterns were recorded to
study the structural properties of the synthesized n-MgO par-
ticles by h–2h method of XRD with a Cu Ka1
(k 5 0.15406 nm) source at 40 kV and 30 mA using multipur-
pose X-ray diffractometer (Bruker D8 Advance, Germany).
The crystalline size of the particles was calculated from the
Debye–Scherrer equation as follows:
0:89k
d5 (2)
bcosh
where, d is the mean size of the ordered (crystalline)
domains, k is the X-ray wavelength, 0.154; b is the line
broadening at half the maximum intensity (full-width at half-
maximum (FWHM)) and h is the Bragg angle.
Environmental Progress & Sustainable Energy (Vol.35, No.3) DOI 10.1002/ep
AFM, SEM, and EDAX Analysis
The morphological characterization of the synthesized n-
MgO was carried out using atomic force microscope (AFM:
Nanosurf, Switzerland; Model: Easy Scan2) and scanning
electron microscope (EVO series-MA15 SEM). EDAX analysis
was done with SEM coupled with EDAX-EVO-MA15-SEM,
Oxford instruments.
Particle Size and Size Distribution
The particle size and its distribution were determined
using particle size analyzer (Horiba scientific SZ-100). A cali-
bration program provided by the instrument manufacturer
was used to calculate the particle size distribution (PSD)
from the attenuation spectra.
Effect of n-MgO on the Growth of Strain Candida Sp
SMN04
To study the effect of n-MgO on the growth of the yeast,
Candida sp. SMN04, the agar well diffusion method was fol-
lowed using various concentrations of n-MgO ranging from 5
to 25 mg mL21. n-MgO was added onto the lawn culture of
the yeast streaked on YEPD agar plate. The test plates were
incubated for 2 days at 308C. A zone of inhibition around the
wells were indicative of growth inhibition. The least concen-
tration, which showed inhibition was taken as a minimal
inhibitory concentration (MIC). The test was supported with
the MIC assay in broth cultures and the cell dry weight was
monitored regularly.
Development of Integrated Nano-Bio System
Integrated nano-bio system was developed following the
method by Li et al. [23]. Yeast cells were grown in YEPD
broth until the mid-exponential growth phase and harvested
by centrifugation at 8400g for 10 min. The cell pellets were
washed twice with phosphate buffer and re-suspended back
in YEPD at a concentration of 2 g L21 of cell dry weight.
About 10 mL of the suspension containing 10 mg of n-MgO
per millilitre of water was mixed with 100 mL of the cell sus-
pension in YEPD broth. The ratio of mass of nanoparticles to
yeast biomass was 2.0 wt/wt. At this ratio, nanoparticles
were sufficient enough to coat the yeast cells. The n-MgO
coated yeast cells termed as nano-bio integrated system was
visualized by AFM and SEM analysis.
Cefdinir Degradation Assay
Batch degradation studies were carried out in 100 mL of
MB containing cefdinir at a final concentration of 250 mg L21
in 250-mL Erlenmeyer flasks incubated on a rotary shaker at
120 rpm and 308C. Degradation experiments employed the
following treatments in MB containing cefdinir, which include
(1) nano-coated yeast cells (integrated nano-bio degradation),
(2) native yeast cells (biodegradation), (3) n-MgO (nanodegra-
dation), and (4) abiotic control. These treatments were used
to compare the efficiency of nano-bio integrated system and
native yeast cells. The experiments were run for 6 days under
aerobic conditions. The supernatant was collected at regular
intervals and residual cefdinir was analyzed by UV-
spectrophotometer (Shimadzu UV-2450), following the
method by Cabri et al. [24] with minor modifications. The
residual cefdinir concentration was calculated using the for-
mula given below,
Ci 2Cf
Residual cefdinir concentrationð%Þ 5 3 100 (3)
C0
Ci is the initial cefdinir concentration and Cf is the final
cefdinir concentration.
May 2016 707
 The obtained degradation data were fitted with pseudo-
first order reaction kinetics [25], for the calculation of half-life
and degradation rate constant using the following equations:
0
Ct 5 C0 : e2k t
(4)
T1=2 5 ln 0:5=2k 0
(5)
where, C0 is the initial concentration of cefdinir in the
medium, Ct is the concentration of cefdinir at time “t”, k0 is
the degradation rate constant, T1/2 is the biodegradation half-
life period of cefdinir.
Enzyme Analysis
To study the enzymatic response of the native and inte-
grated system, activities of various enzymes viz. b-lactamase
[26], cytochrome P450 [27], NADPH reductase [28], amylase
[29], manganese peroxidase [30] were assayed following the
standard protocols by collecting the samples at regular time
intervals. The crude extracts from yeast cells grown in MB
without cefdinir were used as controls. One unit is equivalent
to that amount of enzyme required to catalyze the 1.0 mg of
substrate per minute under standard assay conditions.

RESULTS AND DISCUSSION

Figure 1. X-ray diffraction pattern of n-MgO.
XRD pattern of chemically synthesized magnesium oxide
nanoparticles is shown in Figure 1. The XRD of MgO shows
several diffraction peaks corresponding to 2h values of 36.88,
42.88, 62.28, 74.58, and 78.58 which are indexed as (111),
(200), (220), (311), and (222) diffraction plane of MgO and
crystallizes in cubic structures (JCPDS 45-0946). The peaks
observed in the XRD of MgO are in good agreement with
previous work reported by Mageshwari et al. [31]. The esti-
mated crystalline sizes using Scherrer’s formula for the n-
MgO was found to be 20.4 nm, respectively. The observed
full width half maximum (FWHM) of 0.00726 (in radians)
and h value of 0.373 (in radians) corresponding to the high
intensity peak of the nanomaterial was observed.
The surface morphology of the synthesized nanoparticles
was examined by atomic force microscope (AFM) and scan-
ning electron microscope (SEM). The AFM and SEM micro-
graphs of n-MgO powder is shown in Figures 2a and 2b.

Figure 2. Characterization studies of n-MgO. (a) AFM image; (b) SEM image; (c) histogram showing particle size distribution
and (d) EDAX spectrum. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

708 May 2016 Environmental Progress & Sustainable Energy (Vol.35, No.3) DOI 10.1002/ep
The microscopic studies revealed that, the synthesized mag-
nesium oxide nanoparticles were of irregular shape with
micropores and have a great tendency for agglomeration.
Similar findings were also reported by Athar et al. [22]. The
corresponding particle size distribution of n-MgO revealed
that the average particle diameter of the synthesized n-MgO
was in nanometer range of 10–50 nm, which is in quite
accordance with the reported value [32,33]. The particle size
and distribution of the synthesized n-MgO was evaluated in
solution phase using dynamic light scattering (DLS) tech-
nique. The intensity particle size distribution histogram is
presented in Figure 2c. DLS analyzes the velocity distribution
of particle movement by measuring the dynamic fluctuations
of the light scattering intensity caused by Brownian motion
of the particle. This technique yields a hydrodynamic radius
or diameter, which has to be calculated via Stokes–Einstein
equation from the measurements. It yields an overall mea-
surement of the particle perpendicular to the light source at
Figure 3. Minimal inhibitory concentration (MIC) assay of n-
MgO (mg mL21) on Candida sp. SMN04. [Color figure can
be viewed in the online issue, which is available at wileyon-
linelibrary.com.]
Figure 4. Survival assay of Candida sp. SMN04 grown with n-MgO. (a) Growth curves of Candida sp. SMN04 grown at vari-
ous concentrations of n-MgO. Error bars on the graph represent the standard deviation of triplicate samples (P < 0.001), (b)
survival assay of strain SMN04 grown in the presence of n-MgO at 5 and 10 mg mL21. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
Environmental Progress & Sustainable Energy (Vol.35, No.3) DOI 10.1002/ep
that instant [34]. The SEM particle sizes (10 mm as shown
with scale bar) are larger compared to DLS that may be
attributed to the larger cluster size of aggregated particle
containing a large number of irregular smaller particles.
The elemental analysis of chemically synthesized n-MgO
was done using EDAX spectrum and the results are given in
Figure 2d. It showed a single strong peak at 0.6 keV corre-
sponding to O element (weight percentage: 56.84%) and 1.3
keV corresponding to the Mg element (weight percentage:
43.16%), thereby, confirming the presence of the main ele-
ments in the synthesized nanoparticle which was free from
other impurities. The higher amounts of oxygen weight per-
centage may be due to the interference of surface adsorbed
oxygen during analysis, which might have contributed to the
total oxygen percentage. However, the formation of single
cubic phase of pure MgO was confirmed by powder X-ray
diffraction studies (JCPDS 45-0946) as discussed above.
These results confirmed that the n-MgO formed was in nano-
size and hence these nanoparticles were used as nanocatalyst
for cefdinir degradation in mineral medium.
Effect of n-MgO on growth of Candida sp. SMN04
In general, nanoparticles exert toxic effects on microbes by
disrupting the cell membranes, increasing the membrane per-
meability, interrupting the energy transduction, producing
reactive oxygen species, etc. [17,35]. In order to analyze the
toxicity of n-MgO toward yeast cells, MIC experiment was
conducted (Figure 3). According to MIC results, no zone of
inhibition was noted till 10 mg mL21. Above this concentra-
tion, the yeast cells showed poor or no growth, indicating the
cell damage and microcidal activity of n-MgO with complete
inhibition of cell growth, which was noticed at 15 mg mL21.
Therefore, this concentration was noted as MIC for Candida
sp. SMN04. Hence, the concentration of 10 mg mL21 was
used for the development of integrated nano-bio system for
cefdinir degradation. MIC and survival assay was performed
to determine the lowest concentration of n-MgO that inhibits
the visible growth of the microbe in an overnight culture.
After the preliminary experiments of MIC determination,
survival assay of the yeast cells were demonstrated in YEPD
broth (Figure 4a) and YEPD plate (Figure 4b) containing n-
MgO concentration below its MIC value. The results were
analyzed in terms of cell dry weight and cell growth (g L21)
respectively. Figure 4a explains the growth curve of the yeast
strain with various concentrations of n-MgO ranging from 0
to 15 mg mL21, where the maximum cell dry weight was
May 2016 709
Figure 5. AFM images of Candida sp. SMN04. (a) Native yeast cells; (b) yeast cells grown with 5 mg mL21 of n-MgO; (c) yeast
cells grown with 10 mg mL21 of n-MgO; (d) yeast cells grown with 20 mg mL21 of n-MgO showing topography of each image.
[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 6. SEM Images showing (a) native yeast cell; (b) integrated nano-bio n-MgO coated yeast cells at optimal concentration
of 10 mg mL21 and (c) cefdinir-interacted nano-bio cells recovered from stationary phase.

observed in the medium containing 0 mg mL21 of n-MgO. A
significant amount of biomass was produced in the medium
containing 5 and 10 mg mL21 of n-MgO. When the concen-
tration of n-MgO was increased upto 15 mg mL21, yeast
growth was found to be inhibited (Figure 4a). This result
was supported with the MIC results of our study (Figure 3),
which confirmed that Candida sp. SMN04 could grow in 0,
5, and 10 mg mL21 of n-MgO. Similarly, the growth of the
yeast colonies on YEPD plates also showed that the yeast
cells could tolerate up to 10 mg mL21 of n-MgO. The results
of this experiment were supportive with yeast survival assay
on YEPD plates (Figure 4b). Based on the results, it can be
inferred that the yeast cells could tolerate till 10 mg mL21 n-
MgO without showing any lethal effects or cell damage.
Therefore, 10 mg mL21 concentration of n-MgO was fixed as
an optimum concentration for yeast coating.
Development of Integrated Nano-Bio System
The topography images of AFM of the native cells and
yeast cells with n-MgO (integrated nano-bio system) are pre-
sented in Figures 5a–5d. The native cells showed normal
morphology with oval shaped cells with smooth and intact
cell wall structure (Figure 5a). The AFM images of the nano-
bio integrated system (Figures 5b–5d) showed an increased
surface area due to the coating of n-MgO nanoparticles onto
the surface of the yeast cells. Due to its high surface area, n-
MgO efficiently catalyzed the variety of organic reactions
[36–38]. At lowest concentrations of n-MgO, i.e., 5 mg mL21,
the nanoparticles were attached to the cell surface, and the
yeast showed growing phase with intact cell wall structure
(Figure 5b). On further increasing the concentration of n-
MgO to 10 mg mL21, the yeast cells were still found to be in
growing condition, which indicated that the adsorption of n-
MgO did not show any negative impact on the growth of
yeast cells (Figure 5c). In both cases, the yeast cells were via-
ble in nature as evident from the presence of intact cell
structures. This implied that the concentration of 5 and
10 mg mL21 were not lethal to the yeast strain Candida sp.
SMN04. The yeast cells grown in 20 mg mL21 of n-MgO
showed the absence of proper cell wall structure, which
might be due to the rupture of the cell because of the
increased concentration of n-MgO as shown in Figure 5d.
These observations suggested that the toxic effect of n-MgO
on yeast is directly proportional to the concentration of the
nanoparticles. Thus, it can be concluded that at a concentra-
tion of 10 mg mL21 of n-MgO, the yeast strain SMN04 could
stimulate the coating of n-MgO nanoparticles without caus-
ing any lethal effect to the cubicle. Hence, this concentration
was considered optimum for the development of integrated
nano-bio system, which will induce membrane permeabiliza-
tion to facilitate the entry of pollutant into the cell [39].
Figure 6 shows the scanning electron micrographs of the
surfaces of the native yeast cells and n-MgO-yeast integrated sys-
tem. In Figure 6a, the native cells had normal ovoid cell mor-
phology, whereas, the yeast cells with n-MgO (Figure 6b) clearly
showed that the nanoparticles were efficiently assembled or

710 May 2016 Environmental Progress & Sustainable Energy (Vol.35, No.3) DOI 10.1002/ep
Figure 7. Cefdinir degradation in mineral medium. (a) Residual cefdinir percentage in the culture medium treated with various
degradation agents; and (b) pseudo first-order kinetic plot of cefdinir degradation by various treatments. Error bars on the
curves represent the standard deviation of triplicate samples. [Color figure can be viewed in the online issue, which is available
at wileyonlinelibrary.com.]
Table 1. Comparision of kinetic parameters of cefdinir
degradation in various systems.
Rate constants Half-life; T1/2
Treatments (k day21) (days)
Control 0.0013 533.19
n-MgO 0.1606 4.31
Candida sp. SMN04 0.2335 2.97
Nano-bio integrated 0.4719 1.46
coated onto the surface of the yeast cells by adsorption. In this
condition, the yeast cells were alive and in actively dividing
phase with budding cells. The SEM images revealed that there
were no morphological distortions in case of control and nano-
coated yeast cells. An attractive interaction between the MgO
nanoparticles and microorganisms were reported by other
researchers too [40,41]. Figure 6c showed that the active yeast
cells survived after cefdinir degradation.
Degradation of Cefdinir Using n-MgO and Nano-Bio
Integrated System
Figure 7a compares the degradation percentage of cefdi-
nir in MB with the native yeast cells, n-MgO as well as nano-
coated yeast as nano-bio integrated system. The results
showed that the amount of residual cefdinir was significantly
reduced in the case of nano-bio integrated system. This may
be due to the enhanced entry and synergistic effect of both
nanoparticles and yeast cells toward cefdinir degradation.
Reports on integrated system showing enhanced degradation
than the individual system has been reported by various
workers [19,20,22,42]. The percentage removal of cefdinir
was found to be 84% in case of native yeast cells and 66% in
case of n-MgO particles within 6 days of treatment, whereas,
in case of integrated nano-bio system, 88% removal was
observed within a period of 2.97 days. The faster cefdinir
removal might be due to the synergistic effect of the inte-
grated nano-bio system. However, after the measurement of
residual concentration of cefdinir in various treatments after
10 days, it was found that complete removal of cefdinir was
achieved by integrated nano-bio system (data not shown).
The enhanced entry of cefdinir into the yeast cells occurred
due to the increased permeability as a result of n-MgO coat-
ing on yeast cells, which could positively influence the rate
of cefdinir utilization by the yeast. Shan et al. [42] reported
that the cell–nanoparticles complex generated by assembling
Environmental Progress & Sustainable Energy (Vol.35, No.3) DOI 10.1002/ep
or adsorbing on the surfaces of the microbial cells increases
the reaction activity of original cells and helps in reducing
environmental pollution. Our study reports the complete
removal of cefdinir using integrated nano-bio system with an
initial concentration of 250 mg L21. The increased permeabil-
ity of the yeast cells due to n-MgO coating on their surface
might be another factor, which could have positively influ-
enced faster cefdinir degradation. According to Grigoriev
[39], the association of nanoparticles with microbial cells
facilitates easy transport of pollutant into the intracellular
matrix through a trans-membrane system, leading to the
enhanced degradation of the pollutant.
To understand the reaction kinetics of cefdinir removal,
the obtained degradation data were fitted to pseudo-first
order kinetic model (Figure 7b). The degradation rate con-
stant and half-life were calculated from the kinetic plot and
presented in Table 1. The half-lives of cefdinir degradation
were calculated as 2.97 days and 4.31 days for individual
Candida sp. SMN04 and n-MgO, respectively. Whereas, a
reduced half-life of 1.46 days was obtained in case of inte-
grated nano-bio system. These results suggested that the
degradation of cefdinir by integrated nano-bio system was
found to be more efficient than the individual systems,
which was evidently seen in terms of reduced half-life to
more than half of the value as shown by the individual
yeast and n-MgO.
Enzyme Analysis
The role of degradative enzymes in cefdinir degradation
using individual yeast and integrated nano-bio system were
evaluated through standard enzyme analysis procedures and
the results are presented in Figures 8a and 8b. The antibiotic
activity of cefdinir was tested by growing E. coli in the test
plates where no growth around the well was noted. Cefdinir
degraded products collected at the end of day 1 showed no
inhibitory zone of growth, which confirmed the loss of antibi-
otic property, which demonstrated the involvement of
b-lactamase enzyme during cefdinir degradation (Figure 8a).
This is considered as one of the vital steps in the antibiotic
degradation process [24,43,44]. Similarly, enhanced activities
of other enzymes, viz., cytochrome P450, NADPH reductase,
amylase, and manganese peroxidase were also noted in inte-
grated nano-bio system compared to individual yeast cells
(Figure 8b). This may be due to the synergistic effect of the
integrated system, in which, the nanoparticles might have
stimulated the biotic process. Because of the smaller size and
larger surface area, the nanoparticles was found to improve
May 2016 711
Figure 8. Enzyme assays involved in cefdinir degradation. (a) Demonstration of loss of antibiotic activity (b-lactamase) on day
0 (cefdinir) and day 1 degraded products after degradation; and (b) other degradative enzymes of native Candida sp. SMN04
and integrated nano-bio system. Error bars represent the standard deviation of triplicate samples. [Color figure can be viewed
in the online issue, which is available at wileyonlinelibrary.com.]
the enzymatic activities of microbial cells, which leads to the
release of increased amount of enzymes [45–48]. Additionally,
during scaling up process, the retrieval of MgO nanoparticles
from treated wastewater can be achieved by using commercial
surfactants, which may stop the nanopollution onto the subse-
quent stages of the waste treatment process [49].
CONCLUSIONS
An integrated approach of using n-MgO coated on Candida
sp. SMN04 for enhanced degradation of cefdinir is discussed in
the present study. Experiments conducted in batch mode
revealed that the degradation of cefdinir by integrated nano-bio
system was more competent than the individual systems. The
present work is the first report on the involvement of n-MgO
during cefdinir degradation. The concentration of n-MgO for
coating on the yeast was optimized. The integrated nano-bio
system showed 88% degradation of cefdinir at concentration of
250 mg L21 within two and half days, which was a remarkable
decrease in time compared to the results reported earlier. Fur-
thermore, the involvement of major enzyme, b-lactamase, and
other enzymes was also noted during cefdinir degradation.
Enhanced cefdinir degradation might have occurred through an
integrated approach, which may serve as an effective remedia-
tion tool for the treatment of pharmaceutical wastewater con-
taining cephalosporin antibiotics.
ACKNOWLEDGMENTS
The authors are thankful to the Nanotechnology Lab and
VIT-SAF Lab, SAS, VIT University. They also like to thank
SEM Lab, SBST, VIT University for the instrumental services.
The financial support and laboratory facility provided by VIT
University, Vellore are duly acknowledged.
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