Mechanistic investigations in ultrasound-assisted biodegradation of phenanthrene

Article abstract of DOI:10.1016/j.ultsonch.2019.104890

This study has addressed the biodegradation of polycyclic aromatic hydrocarbon, phenanthrene using Candida tropicalis. Optimization using central composite statistical design yielded optimum experimental parameters as: pH = 6.2, temperature = 33.4 °C, mechanical shaking = 190 rpm and % inoculum = 9.26% v/v. Sonication of biodegradation mixture at 33 kHz and 10% duty cycle in log phase (12 h per day for 4 days) resulted in a 25% enhancement in phenanthrene removal. Profiles of specific growth rate (μ) and specific degradation rate (q) versus initial substrate concentration were fitted to Haldane substrate inhibition model. Both μ and q showed maxima for initial concentration of 100 mg L^?1. Kinetic analysis of degradation profiles showed higher biomass yield coefficient and smaller decay coefficient in presence of sonication. Expression of total intracellular proteins in control and test experiments were analyzed using SDS–PAGE. This analysis revealed overexpression of enzyme catechol 2,3-dioxygenase (in meta route metabolism) during sonication which is involved in ring cleavage of phenanthrene. Evaluation of cell viability after sonication by flow cytometry analysis revealed > 80% live cells. These effects are attributed to enhanced cellular transport induced by intense microturbulence generated by sonication.

Full text of DOI:10.1016/j.ultsonch.2019.104890

Journal Pre-proofs
Mechanistic investigations in ultrasound–assisted biodegradation of phenan-
threne
Niharika Kashyap, Kuldeep Roy, Vijayanand S. Moholkar
PII:
S1350-4177(19)31572-X
DOI:
https://doi.org/10.1016/j.ultsonch.2019.104890
ULTSON 104890
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Ultrasonics Sonochemistry
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Revised Date:
Accepted Date:
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18 November 2019
19 November 2019
Please cite this article as: N. Kashyap, K. Roy, V.S. Moholkar, Mechanistic investigations in ultrasound–assisted
biodegradation of phenanthrene, Ultrasonics Sonochemistry (2019), doi: https://doi.org/10.1016/j.ultsonch.
2
019.104890
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Mechanistic investigations in ultrasound–assisted biodegradation of
phenanthrene
1
2
1,2,*
Niharika Kashyap , Kuldeep Roy , and Vijayanand S. Moholkar
1
2
Centre for the Environment, Department of Chemical Engineering
Indian Institute of Technology Guwahati
Guwahati – 781 039, Assam, India
*
Author for correspondence. E–mail: vmoholkar@iitg.ac.in, Fax: +91 361 258 2291
Abstract
This study has addressed the biodegradation of polycyclic aromatic hydrocarbon, phenanthrene
using Candida tropicalis. Optimization using central composite statistical design yielded optimum
experimental parameters as: pH = 6.2, temperature = 33.4C, mechanical shaking = 190 rpm and %
inoculum = 9.26% v/v. Sonication of biodegradation mixture at 33 kHz and 10% duty cycle in log
phase (12 h per day for 4 days) resulted in a 25% enhancement in phenanthrene removal. Profiles
of specific growth rate (µ) and specific degradation rate (q) versus initial substrate concentration
were fitted to Haldane substrate inhibition model. Both µ and q showed maxima for initial
–
1
concentration of 100 mg L . Kinetic analysis of degradation profiles showed higher biomass yield
coefficient and smaller decay coefficient in presence of sonication. Expression of total intracellular
proteins in control and test experiments were analyzed using SDS–PAGE. This analysis revealed
overexpression of enzyme catechol 2,3–dioxygenase (in meta route metabolism) during sonication
which is involved in ring cleavage of phenanthrene. Evaluation of cell viability after sonication by
flow cytometry analysis revealed >80% live cells. These effects are attributed to enhanced cellular
transport induced by intense microturbulence generated by sonication.
Keywords: Polycyclic aromatic hydrocarbons; Candida tropicalis; Phenanthrene; Biodegradation;
Haldane kinetic model; Ultrasound
1

1
.
Introduction
Polycyclic aromatic hydrocarbons (PAHs) appearing in discharges from chemical and process
industries, have gained noteworthy attention for decades due their extraordinary stability in
terrestrial/aquatic environments, resistance to degradation through conventional advanced
oxidation processes (AOPs) and harmful effects on ecosystem [1], [2]. Bioremediation of PAHs has
emerged as a potential alternative to conventional oxidative processes. Several ligninolytic or non–
ligninolytic fungi have been employed for bioremediation [3], [4], [5], [6]. However, for
bioremediation of wastewater, non–ligninolytic fungi are more suitable, since the ligninolytic fungi
have habitat limitation of growth [7]. The most commonly used non–ligninolytic fungi for
bioremediation of PAH belong to genera Candida, Fusarium, Aspergillus, Penicillium, and Pichia
[8], [9], [10]. Microbial degradation route of PAHs by non–ligninolytic fungi normally comprises
of three phases, viz. (a) activation of aromatic ring, (b) dearomatization, (c) transformation of ring
fission products into tricarboxylic acid (TCA) cycle metabolites. A normal pathway of metabolic
cleavage of PAH comprises of dihydroxylation of benzene ring into catechol derivative and then
ring fission via ortho (intradiol) or meta (extradiol) oxidation route. Catechol undergoes oxidation
either by cleavage enzyme catechol 1,2– dioxygenase (C1,2–D) to form cis, cis–muconic acid
through ortho– route. Aletrantively, catechol may also be oxidised by cleavage enzyme catechol
2
,3–dioxygenase (C2,3–D) through meta– route resulting in formation of 2–hydroxymuconic
semialdehyde (HMSA) [11]. A brief review of representative literature on degradation of different
PAHs is provided in Table 1. It could be inferred that the degradation kinetics of PAHs by different
microbial strains is quite slow. Moreover, many yeasts need to be supplemented with additional
carbon source, surfactants and other additives such as yeast extract.
The degrading capability of microorganisms also depends on physical parameters like pH,
carbon sources, temperature, and nutrients [12]. Biodegradation of PAHs can be enhanced by proper
optimization of the physical process parameters [13], [14]. Bioremediation can also be coupled with
other techniques of process intensification. One of the contemporary techniques, for enhancing
2

kinetics/yields of biochemical processes is ultrasound irradiation or sonication [15]. Previous
authors have coupled sonication with inorganic catalysts for boosting the kinetics of degradation of
organic pollutants [16], [17]. Sonication of moderate intensity is known to augment the kinetics of
the bioprocesses. Physical and chemical effects of sonication are responsible for intensification of
the process. The phenomenon of cavitation (growth, nucleation, and the transient collapse of
bubble) is responsible for the physical/chemical effects of sonication. The major physical effect
produced by sonication is intense microturbulence in the system, and the chemical effect is
formation of highly reactive radicals through thermal dissociation of gas and vapor molecules in
the cavitation bubble.
In this study, we have attempted to investigate ultrasound–assisted biodegradation of PAHs
from a mechanistic viewpoint. The model system of present study comprises of phenanthrene and
Candida tropicalis MTCC 184. The present study begins with statistical optimization of
degradation parameters followed by experiments on ultrasound–assisted degradation at optimum
conditions. The experimental profiles of biomass and substrate during bioremediation have been
analysed using kinetic models that account for substrate inhibition. The predominant metabolic
pathway of phenanthrene degradation has been identified in mechanical shaking (control) and with
sonication (test). Moreover, over–expression of cellular proteins during sonication were also
assessed using SDS–PAGE analysis. Concurrent analysis of these studies has revealed interesting
mechanistic aspects of ultrasound–induced enhancement of phenanthrene biodegradation by C.
tropicalis cells. Finally, the effect of sonication on cell viability and morphology has been assessed.
2
2
.
Materials and methods
.1.
Microbial growth and maintenance
Yeast strain, C. tropicalis MTCC 184, was acquired from the Institute of Microbial
Technology (IMTECH), Chandigarh, India. The yeast strain was conserved by sub–culturing on
yeast extract peptone dextrose (YEPD) agar plates and storage at 4C. Sub–culture was done every
3

month and preserved at –80C in glycerol (20% v/v). The culture was grown at 30C, 150 rpm
agitation, and pH 7. For PAH biodegradation, the yeast was grown in Bushnell–Hass (BH) minimal
–
1
salt medium (MSM) with a composition of (g L ): K HPO (1.0); KH PO (1.0); NH NO (1.0);
2
4
2
4
4
3
FeCl (0.05); MgSO ·7H O (0.2) and CaCl 2H O (0.02). The biodegradation experiments were
3
4
2
2
2
performed in BH medium with phenanthrene (PAH) as sole carbon source.
2
.2.
Chemicals, medium, and reagents
Phenanthrene (sublimed grade, ≥ 99.5%) was procured from Sigma–Aldrich (India).
Bushnell Haas medium (BHM) was procured from Himedia (Mumbai, India). HPLC grade
acetonitrile, acetone, and n–hexane were purchased from Merck, India. The rest of the chemicals
(analytical grade) used for analysis were acquired from Himedia (Mumbai, India).
2
.3.
Biodegradation experiments
In a typical experiment, the yeast culture was initially grown in YEPD medium at 30C, 150
rpm agitation and pH 7. The pre–culture was incubated until it attained an optical density of 0.5 
.6 at 600 nm (OD600 nm). Successively, yeast biomass was separated through centrifugation,
0
washed, and the pellets were suspended in BH medium. Next, the pre–culture was inoculated in the
degradation medium with phenanthrene (PAH) as the sole carbon source. Prior to degradation, BH
medium was sterilized (autoclaved at 121°C, 20 min) followed by inoculation of seed culture.
Phenanthrene has negligible solubility in pure water. Hence, 10 mg phenanthrene was dissolved in
minimum acetone (2  3 mL), and this mixture was added to working volume of 100 mL BHM.
–
1
Thus, the phenanthrene concentration in resulting solution is ~ 100 mg L . Un–inoculated controls
flasks containing only BHM and phenanthrene were maintained to validate any abiotic loss. The
experiments were carried out for 14 days and samples of the reaction mixture were collected at
regular intervals to monitor cell biomass growth and residual phenanthrene.
4

After completion of 14-day experiment, samples were initially extracted using an equal
volume of n–hexane. The mixtures were vortex agitated, followed by 10 min centrifugation (10,000
rpm) to transfer residual phenanthrene in the organic layer. The solution was allowed to settle, and
the organic layer comprising n–hexane, and phenanthrene was procured in vials. Samples with three
replicates were prepared and were analyzed using high performance liquid chromatography (HPLC,
Agilent Technologies, 1220 Infinity LC) fitted with C18 reverse–phase column (3 mm 150 mm
and particle size of 3.5 m). Mixture of acetonitrile: milli–Q water (volume ratio 70:30) was used
as the mobile phase (or eluent). The sample injection volume was 20 µL and flow rate of mobile
−
1
phase was 0.8 mL min flow rate. Phenanthrene was identified with a UV detector (254 nm), and
retention time was compared against standards of phenanthrene.
2
.4.
Statistical optimization of phenanthrene degradation
The physical parameters related to phenanthrene biodegradation were optimized by
response surface methodology using the central composite design of experiments. Four
experimental parameters, viz. medium pH, % inoculum, temperature, and mechanical shaking, were
taken as the independent variables (or optimization parameters). Percentage degradation of
phenanthrene was the response (or dependent) variable. The notations for optimization parameters
(
or factors) and their levels (with actual and coded values) are provided in Table 2. The complete
design of 31 experiments (MINITAB, Trial version) was based on five coded levels (–, –1, 0, +1,
) of optimization parameters. This is provided in supplementary material. The results of
+
statistical experimental design were analysed using response surface methodology.
The experimental data acquired from central composite design (provided in supplementary
material) was fitted to the quadratic equation with individual and interactive coefficients
corresponding to optimization parameters:
k
k
2
i
Y   
 X 
 X 
 X X
 ij
(1)
0
i1
i
i
i1 ii
i
j
5

Notation:
Y
 i ii
− dependent variable (% PAH degraded), k − number of factors,  0 , , , −
ij
regression coefficient acquired for constant, linear, quadratic, and interaction, respectively. The
coded values (X) of the optimization parameters are defined as: X  xi  x0

x, where i = 1, 2,
3
…, xi is a dimensionless value of the variables, x0 is the value of xi at center point, and x is
the step change.
Validation (or control) experiments: For validation of statistical optimization, phenanthrene
biodegradation experiments were conducted in triplicate at optimum conditions (or parameters)
predicted by the statistical experiments. The time profiles of biomass and substrate concentration
were analyzed with a kinetic model (described in subsequent sections).
2
.5.
Phenanthrene tolerance of C. tropicalis:
The tolerance of C. tropicalis strain towards phenanthrene was examined with two
important factors, viz. (1) biomass growth pattern, and (2) degradation potential of the strain under
–
1
different initial phenanthrene concentrations in the range 0  500 mg L . 100 mL BH medium
supplemented with phenanthrene (at different initial concentrations) was inoculated with preculture
of C. tropicalis cells. All experiments were done at optimum conditions obtained from statistical
design. A control experiment was done to assess the growth of C. tropicalis in BH medium in
absence of phenanthrene or any other carbon source. All experiments including the control were
conducted for 14 days with three times for the assessment of reproducibility.
2
.6.
Ultrasound–assisted biodegradation of phenanthrene (test experiments)
Ultrasound–assisted biodegradation of phenanthrene was conducted in an ultrasonic bath (PCi
Analytics, India, dimensions  30 cm × 15 cm × 10 cm, volume  3.5 L, rated power 100 W,
frequency 33 kHz). Before experiments, ultrasonic bath was calibrated with calorimetric technique
to determine the ultrasound pressure amplitude and the actual power input. The pressure amplitude
6

of ultrasonic waves in the medium corresponding to calorimetrically measured acoustic intensity of
–
2
0
2
.66 W cm was 1.4 bar (or 140 kPa). The water temperature in ultrasound bath was kept at 32° ±
°C. Moreover, location of the Erlenmeyer flask in the bath was maintained same in every
experiment to prevent artifacts due to spatial variation of ultrasound intensity [18].
In biodegradation test experiments, all physical parameters (except mechanical shaking) were
at their optimum values, as obtained in validation experiments. Mechanical shaking in control
experiments was replaced with a combination of mechanical shaking and sonication at optimum
duty cycle. The duty cycle of sonication was also optimized before experiments. Optimum results
were obtained for 10% duty cycle, i.e. sonication  1 min and mechanical shaking  9 min for every
1
0 min reaction. Sonication was applied to the broth during log phase of yeast cells (total four days
from day 4 to day 8) for 12 h/day @ 10% duty cycle. For the remaining 12 h/day of log phase, the
reaction mixture was subjected to mechanical shaking (as done in validation experiments). The
experimental protocol of control (mechanical shaking) and test (mechanical shaking + sonication
@
10% duty cycle) experiments is explained in Fig. 1. The duration of all biodegradation
experiments was 14 days.
2
.7.
Analysis of the viability and morphology of yeast cells exposed to sonication
Viability of ultrasoundexposed C. tropicalis MTCC 184 cells in test experiments was
analyzed by Flow cytometry using a multi–parametric BD FACS Calibur (Becton Dickson,
CaliburTM Flow Cytometer, USA). Microbial cells after completion of experiment were examined
with 488 nm laser and 530 nm emission filter using propidium iodide (PI). The changes in cell
morphology during sonication were assessed on the basis of SSC (Side Scatter) and FSC (Forward
Scatter). Side–scattered light (SSC) is proportional to cell complexity or granularity, and forward–
scattered light (FSC) is proportional to cell size or surface area.
The C. tropicalis cells in both control and test experiments were analysed with FE–SEM
(Field Emission Scanning Electron Microscope, Make: Zeiss, Model: Sigma 300) micrographs to
7

spot any visible differences in morphological and phenotypic characters. Initially, the cells were
centrifuged for 10 min at 5,000 rpm, and the resultant pellet was washed thrice with phosphate
buffer saline (PBS, pH 7). Further, the pellet was fixed with 2.5% formaldehyde for 1 h and again
washed thrice with PBS, followed by a dehydration step using ethanol at different concentration
range (10, 30, 50. 70, 90, 100% ethanol) for 30 min. At last, the samples were drop cast on the FE–
SEM grid and allowed to dry at 37C. The grids were coated with gold powder and observed under
FE–SEM at magnification of 2.00 KX.
2
.8.
Chemical oxygen demand (COD) measurement and FTIR analysis
The extent of phenanthrene biodegradation was also assessed in terms of reduction in
chemical oxygen demand (COD) and analysis of Fourier Transform Infrared (FTIR) spectra of the
samples. Samples from control and test experiments were centrifuged for 5 min at 10,000 rpm, and
the aqueous supernatant was taken for COD and FTIR analysis. COD of the samples was quantified
using HACH COD reagents and measured in DR900 calorimeter (Hach, USA). The percentage
reduction of COD was calculated from initial COD and final COD values.
The absorption bands in the FTIR spectra correspond to the functional groups present in
molecular species formed out of phenanthrene biodegradation. The samples were examined after
lyophilization (Labconco) using FTIR (Perkin Elmer Spectrum two), and the resulting spectra were
matched with FTIR spectrum of pure phenanthrene. The FTIR analysis was done in mid–infrared
−
1
region from 400 to 4000 cm .
3
.
Kinetic analysis of phenanthrene biodegradation
The previous literature [19] has reported significant substrate inhibition effects during the
growth of yeasts, such as C. tropicalis on organic pollutants. Substrate (phenanthrene) utilization
rate is written as:
8

dS
dt

qX    Y


X
(2)
S
= phenanthrene concentration (mg L ), µ = specific biomass (or cell)
x/ s
–
1
Notations are as follows:
–
1
= incubation time (day),
X
= dry cell weight (mg L–1), Yx s = growth yield
growth rate (day ),
t
–
1
–1
coefficient (g g ),
q
= specific degradation rate (day ). Yield factor for biomass,
Y
(g dry
x/s
biomass/g phenanthrene) can be calculated as follows:
dX dt
dS dt
dX
dS

X  X0

Yx s  
 
 
(3)

S  S0

Profile of biomass involving growth and decay is written as:
dX

X bX
(4)
dt
–
1
where
b
= biomass decay coefficient (day ). For the integration of equations 2 and 4, we have
used the value of specific growth rate (µ) corresponding to initial substrate concentration (S = S0).
Several kinetic models that account for substrate inhibition are available in the literature. The basic
kinetic model for substrate inhibition is Haldane model:
max
S


(5)
2
K  S  S K


S
i
SS0
–
1
–1
where, S = phenanthrene concentration (mg L ), µ = specific biomass growth rate (day ), 
=
max
–
1
–1
maximum specific biomass growth rate (day ), K = half saturation coefficient (mg L ), K =
s
i
–
1
inhibition coefficient (mg L ). However, other semi–empirical models reported in the literature
are:
max
S
Aiba model:
(6)
(7)


exp

 S Ki  SS
0
K  S
s

2
S
max
Edward model:


S  K  S K 1 S Ks



 SS
s
i
0
9


S
max
2
Yano model:  
(8)
S  K  S K 1 S K



 SS
s
i
0
The kinetic parameters in the above models were determined from the analysis of µ and q over a
–
1
wide range of initial substrate concentrations, viz. S = 25 to 500 mg L . The specific biomass
0
growth rate (µ) in the log or exponential phase of each experiment was obtained using basic relation:
dX dt   X (i.e., equation 4 with decay coefficient assumed as zero), or as slope of the plot
ln

X Xi

versus t. The plot of µ versus S was fitted to equations 5 to 8 using non–linear regression
0
method in MATLAB R2018b to yield the growth coefficients µmax, K, K and K . The best model
S
i
for the analysis was selected on the basis of regression coefficient. For determination of specific
degradation rate (q), a similar procedure was followed, in which the experimental values of q
(
specific degradation rate) for varying initial substrate concentrations (S ) were determined from
0
the degradation profiles for the total 14day period of experiment. This data was fitted to analogous
expressions for q (as a function of S at S = S ), as given in equations 5 to 8, to obtain parameters of
0
qmax, .
K, K, K
i
S
The kinetic parameters of b (decay coefficient) and YX/S (biomass yield coefficient) in eqs. 2
and 4 were obtained by fitting experimental profiles of biomass and substrate to eqs. 2 and 4 using
Genetic Algorithm in MATLAB (R2018b). These equations were simultaneously solved using
th
Runge–Kutta 4 order method (as initial value problem) to obtain the simulated profiles of substrate
and biomass. The simulated profiles were compared against the experimental profiles (mean values
of biomass and substrate concentrations in the triplicate experimental runs). The sum of RMSE
(root mean square error) between the two profiles was minimized by adjusting the numerical values
of kinetic parameters in the model within specified bounds [20]. For this purpose, experiments (both
control and test) were conducted at optimum conditions obtained from statistical experiments. The
initial substrate concentration (corresponding to maximum q and µ) for these experiments was
obtained from analysis of substrate inhibition kinetics outlined above.
1
0

4
.
Protein expression and identification of metabolic pathway
The expression and size of crude proteins present in the samples were established by running
SDS–PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis). Moreover, phenanthrene
degradation pathway by C. tropicalis (either ortho– or meta–cleavage route) under test and control
conditions was also identified. The principal enzymes involved in the pathway, viz. catechol 1,2–
dioxygenase (C1,2–D) and catechol 2,3–dioxygenase (C2,3–D), are responsible for catechol ring
fission through ortho or meta route. The predominant degradation pathway can be identified by
conducting an assay of catechol dioxygenase enzyme activity.
4
.1.
Protein extraction and expression (SDS–PAGE analysis)
Samples of broth (control and test experiments) were collected and C. tropicalis cells were
separated by centrifugation at 10,000 rpm for 5 min. The resulting cell pellets were suspended in
lysis buffer (5% SDS, 8 M urea, 0.1 mM EDTA, 40 mM Tris–HCl pH 7.5) [21]. Further, the cells
were ruptured using a sonicator probe (Sonics & Materials VCX 500) @ 50% duty cycle (30 s on
and 30 s off) to extract intracellular proteins. The samples were further centrifuged for 30 min at
1
0,000 rpm and at 4C and supernatant were separated. To confirm the expression of proteins, SDS–
PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis was done. 40 µL of
the sample was mixed with SDS loading buffer and reducing agent, and then resolved on a 12%
SDS–polyacrylamide gel (PAGE). The SDS gel was stained with coomassie brilliant blue dye and
was observed in Gel Doc (Bio–RAD, USA) with Image Lab 5.2.1 software. The protein bands were
examined according to their respective molecular weights. The total protein content present in
samples was estimated by Lowry method with bovine serum albumin (BSA) as standard [22].
4
.2.
Analysis of enzyme activity
1
1

Activities of the two enzymes in the metabolic pathway, C1,2–D, and C2,3–D, were
th
th
assessed by growing C. tropicalis in BHM with phenanthrene as sole carbon source. 7 and 14 –
day samples of control and test experiments were used for enzyme activity assay. Catechol
dioxygenase activity in the extract was determined according to the standard procedure [23].
4
.3.
Analysis of catechol 1,2–dioxygenase activity
The catechol 1,2–dioxygenase activity is a signature of the ortho–cleavage reaction of
catechol leading to cis,cis–muconic acid formation. This activity can be quantified using
spectrophotometric measurements. The reaction mixture for assessment of C1,2–D activity was
added in a quartz cuvette with following composition (total 3 mL): 0.1 mL 2–mercaptoethanol (100
mM), 0.7 mL distilled water, 2 mL Tris–HCl buffer (50 mM, pH 8), 0.1 mL cell–free sample. After
thorough mixing, 0.1 mL catechol (0.1 mM) was added. The absorbance of this solution at 260 nm
was monitored for 5 min. The rise in absorbance of solution was an indicator of cis–cis muconic
acid production [24].
4
.4.
Analysis of catechol 2,3–dioxygenase activity
The presence of catechol 2,3–dioxygenase activity signifies the meta–cleavage reaction of
catechol with the formation of 2–hydroxymuconic semialdehyde (HMSA). The reaction mixture
for assessment of the activity consisted of 0.2 mL cell–free sample, 2 mL Tris–HCl buffer (pH 7.5),
and 0.6 mL distilled water. Catechol (100 mM) was added after thorough mixing, and then
absorbance was monitored at 375 nm for 5 min. A rise in absorbance essentially signifies formation
of HMSA [24].
4
.5.
Calculation of enzyme activity
1
2

The enzymatic activity of one unit (U) can be defined as the quantity of enzyme converting
one μmol of the substrate into the product in one minute under specific reaction conditions and can
be estimated as:

E V 1000
-
1
f
Activity (U mL ) 
(9)

t V  L E
s
m
where, ΔE = change in absorbance, V = final volume, V = cell–free extract volume, Δt = time of
f
s
reaction (5 min), E = molar extinction coefficient of the formed product and L = path length (1 cm
m
for standard cuvette). Specific activities of the samples were expressed as units per milligram of
protein.
5
5
.
Results and Discussion
.1.
Statistical optimization of process parameters
nd
The experimental data obtained from central composite design was fitted to a 2 –order
regression equation as:
Y  45.857  0.812X  4.895X 1.312X  3.645X 9.792X  6.354X
2
2
1
2
3
4
1
2
2
3
2
4

0.354X  4.792X  2.406X X  0.031X X  2.343X X  0.218X X
(10)
1
2
1
3
1
4
2
3

0.656X X 1.593X X
2
4
3
4
The full CCD experimental design, along with the predicted and experimental values of response
variable (i.e. percentage degradation) is provided in supplementary material. The global optimum
values of variables (process parameters) corresponding to maximum response variable
(phenanthrene degradation) were obtained as: medium pH = 6.2, temperature = 33.4°C, agitation
speed = 190 rpm and % inoculum = 9.26 vol%. At optimum conditions, the maximum phenanthrene
degradation predicted by the quadratic model was 65.19%.
Validation experiment: The substrate and biomass profiles of the validation experiment are given
in supplementary material. For 100 mg L–1 initial phenanthrene concentration, a residual
concentration of 34  2 mg L–1 was obtained after 14 days, which corresponded to ~ 66%
1
3

degradation. This result matched closely with the predictions of statistical experimental design.
5
.2.
Tolerance of C. tropicalis to phenanthrene
The tolerance of the yeast strain to phenanthrene was studied by performing batch
experiments with different initial concentrations of phenanthrene (at optimum conditions predicted
by statistical design). The trends of final biomass concentrations and residual phenanthrene
concentration are given in supplementary material. The final biomass concentration in the mixture
–
1
shows a maximum for initial concentration of 100 mg L and reduces thereafter. This result
demonstrates toxicity of phenanthrene towards the growth of C. tropicalis cells. The absolute
phenanthrene degradation rises with initial concentration in the mixture, but not proportionately,
and this could be a manifestation of inhibition of biomass growth by phenanthrene.
5
.3.
Kinetic analysis of growth and phenanthrene degradation
The profiles of µ (specific growth rate) and q (specific degradation rate) for different initial
concentrations of phenanthrene (S ) are shown in Fig. 2. These profiles were fitted to various
0
substrate inhibition models using nonlinear regression method (MATLAB R2018b). The kinetic
parameters in various models obtained after fitting the profiles are listed in Table 3. The Haldane
2
model had the best fit for profiles of both µ and q with the highest R and the lowest RMSE. The
–
1
kinetic parameters for Haldane model for biomass growth were: µmax = 0.6 day , K = 27.54 mg
s
–
1
–1
L , K = 428.4 mg L . The corresponding parameters for degradation profile were: q = 0.18
i
max
day , Ks = 46.89 mg L–1, Ki = 117.5 mg L . The profiles of µ and q in Fig. 2 show maxima at S
–
1
–1
0
–
1
–1
=
100 mg L . Reduction in µ and q after S = 100 mg L is an indication of substrate inhibition at
0
relatively higher concentrations of phenanthrene. Moreover, relatively faster reduction in q with
initial substrate concentration after S = 100 mg L–1 indicates greater influence of substrate
0
inhibition on phenanthrene degradation than biomass growth. This is also reflected in terms of ~4×
1
4

higher value of K (inhibition constant for biomass growth) than Ki (inhibition constant for
i
substrate degradation).
5
.4.
Phenanthrene degradation: control and test experiments
The results of phenanthrene degradation under control (mechanical shaking), and test
(combination of sonication @ 10% duty cycle and mechanical shaking) conditions are shown in
Fig. 3. The experimental time profiles of concentrations (substrate and biomass) were fitted to
equations 2 and 4 using numerical solutions coupled with Genetic algorithm. The simulated time
2
profiles of biomass and substrate are also shown in Fig. 3. The regressions coefficients R for all
profiles are > 0.9, indicating good match between simulations and experimental profiles. The final
results of both experiments (control and test) are tabulated in Table 4, which depicts the kinetic
parameters (b and Y ) in eqs. 2 and 4, along with total absolute phenanthrene degradation. The
X/S
phenanthrene degradation shows ~ 25% rise under test conditions. The biomass yield coefficient
(YX/S) also shows 25% rise in test experiments, while the decay coefficient (b) reduces ~ 21%.
Enhanced phenanthrene degradation with higher cell growth in test experiments can be attributed
to several beneficial effects of sonication on reaction system as follows:
(
1) intense microturbulence generated by sonication enhances the cell membrane permeability,
which facilitates quicker cellular transport of nutrients, substrate, and other metabolites [25], [26].
2) microturbulence also causes deagglomeration of cell clusters, which helps better access to
nutrients and substrate in broth [27].
3) microturbulence also causes dilution of toxic substances produced during substrate utilization,
(
(
which helps in reducing cell death and enhancing cell growth.
In the present study, we have attempted to explore an additional facet of protein
overexpression induced by sonication that could influence intracellular metabolism, as explained in
subsequent section.
1
5

5
.5.
Viability and morphological changes in yeast cells exposed to sonication
Fig. 4 demonstrates the results of flow cytometry study for assessing the viability of C.
tropicalis cells after exposure to ultrasound. Live and dead yeast cells were characterized by their
distinct staining characteristics with PI stain. No substantial alteration occurred in FSC and SSC of
the cells after sonication, as shown in Figs. 4 A and B, which implied that the cell morphology and
internal complexity of C. tropicalis remained practically unchanged after sonication. The
percentage of live cells present in control and test experiments was determined from the acquisition
dot plots between FSC and FL3 given in Figs. 4 C and D. The percentage of live cells in both test
(
82.80%) and control (84.93%) experiments did not differ much. In concurrence, the histograms of
PI + cells in Figs. 4 E and F show a similar percentage of dead cells in control (14.93%) and test
17.04%) experiments. These results clearly demonstrate no adverse effect of sonication on
microbial cells.
The FESEM micrographs of C. tropicalis cells are shown in Figs. 5A and B. A comparison
(
of these micrographs does not show any significant difference in morphology and topography of
the cells. The micrographs for test experiment also do not show any visible cell damage, rupture, or
debris, indicating that the cells remained intact during exposure to sonication. Thus, the results of
FESEM analysis were in concurrence with the results of flow cytometry.
5
.6.
COD removal and FTIR analysis
Chemical oxygen demand (COD) was used as an indicative measure of residual organic
matter. The reduction in COD in control and test experiments was monitored. It was observed that
0% COD removal was achieved in control experiments, while 69% COD removal was achieved
6
in test experiments. Relatively higher reduction in COD is attributed to enhancement of
phenanthrene biodegradation by sonication. Moreover, only 3  5 % of COD was degraded in the
abiotic samples. Another probable cause of higher COD removal by sonication is formation of
●
watersoluble products due to reaction between phenanthrene and OH radicals generated by
1
6

sonication. Greater details of COD analysis are given supplementary material.
FTIR analysis was used to identify the functional groups of the species present in samples of
both experiments (control and test), which are indicative of phenanthrene degradation. The IR
−
1
spectra of the samples are shown in Fig. 6. In Fig. 6 A, the band between 2800  3200 cm (3054
-1
cm ) is resultant of proton (H) stretching and indicates aromatic C-H band stretching in pure
-1
-1
-1
-1
phenanthrene [28]. Similarly, bands between 1200  1600 cm (1451 cm , 1454 cm , 1498 cm )
denote the skeleton vibration peaks of benzene ring in pure phenanthrene. Moreover, band at 729
−
1
−1
cm signify four binding proton connected to benzene ring and the band at 815 cm denotes a
further proton bond to benzene ring. The spectrum of final sample (drawn at the end of experiment)
is different from the spectrum of pure phenanthrene, as it contains several intermediate species
formed during biodegradation of phenanthrene. In control and test experiments (Fig 6 B and C),
-1
-1
intense peaks were observed at 3262 cm and 3257 cm , respectively, signifying free O-H
-1
-1
stretching vibrations, while strong bands at 1595 cm and 1634cm correspond to aromatic CC
−
1
stretching and NH bending vibration. The peaks within 1039 – 1400 cm range corresponds to
2
-1
CH , CH bending deformation, and CN stretching, while peaks in the range of 800 cm and 500
3
2
−
1
cm indicate CH bend and CBr stretch, respectively. The FTIR results thus confirm degradation
of phenanthrene with formation of different metabolites having different functional groups. It is
noteworthy that FTIR spectra of final samples from both experiments (control and test) comprise
of peaks in the same range, indicating formation of same metabolites (or in other words same
degradation pathway). This also indicates that sonication only enhances the kinetics of metabolic
reactions but does not alter the metabolic pathway.
As seen from Table 1, the phenanthrene degradation (84% in 14 days) as well as COD
removal (69%) in the present work was higher than previous studies employing fungi and yeast (or
specifically the Candida sp.). Moreover, a peculiar facet of the present study, as compared to the
previous literature, is that phenanthrene has been used as sole carbon source with no
1
7

supplementation of other carbon source or additives.
5
.7.
SDS–PAGE analysis of protein expressions
The intracellular proteins in the samples from control and test experiments were extracted
and resolved on a 12% SDS–polyacrylamide gel (PAGE) by electrophoresis and stained with
coomassie brilliant blue. The protein ladder marker used in the experiment was protein molecular
weight marker (Low) (97.2 kDa to 14.3 kDa) (Company: Takara, Code no:3450) [29]. Fig. 7 shows
the gel image of SDS–PAGE analysis of protein expression in control and test experiments.
Comparing the protein bands in lane 1 (test) and lane 2 (control) reveals much higher expression of
the proteins in test experiments. This is clear evidence of enhanced metabolism that results in faster
degradation of phenanthrene.
Bands of different sizes were visible in the lanes of both control and test samples. Among
various proteins that facilitate phenanthrene degradation, catechol 2,3–dioxygenase (C2,3–D),
possessing a size of 35.0 kDa [30], is a key enzyme in the meta–route of degradation (refer to Fig.
8
depicting the metabolic pathway of phenanthrene degradation). SDS–PAGE analysis of both test
and control samples revealed that it contained a protein band (Fig. 7), whose size was in good
agreement with that of C2,3–D (35.0 kDa). Moreover, this band was more expressed in test samples
(lane 1), indicating higher production of this protein under the influence of sonication [31].
5
.8.
Identification of metabolic pathway
Biodegradation of phenanthrene, as depicted in Fig. 8, is known to occur via catechol
degradation pathway. Catechol is a common intermediate in polycyclic aromatic biodegradation
24], [32]. It is further degraded through ortho– or meta– cleavage route, as displayed in Fig. 8. As
[
noted earlier, the ortho–cleavage route leads to cis,cis–muconic acid, whereas the meta route results
in HMSA formation. Contributions of both meta and ortho pathways can be discerned by
quantification of the characteristic enzymes, C2,3–D for meta pathway, and C1,2–D for ortho
1
8

pathway [23], [33]. To identify the impact of sonication on catechol ring cleavage route, enzymatic
th
th
assay for C1,2–D and C2,3–D was carried out on 7 and 14 day of degradation in both control and
test experiments. There was no rise in absorbance at 260 nm during C1,2–D assay in control and
test experiments, i.e. no C1,2–D activity and no cis,cis–muconic acid production. During C2,3–D
th
th
assay for 7 and 14 day of degradation, rise in absorbance at 375 nm was observed in both control
and test experiments signifying strong activity of C2,3–D enzyme. Absorbance profiles of C2,3–D
enzyme in control and test experiments are given in supplementary material. The absorbance values
for test experiments were higher than control for both days, which indicates greater enzyme
production and faster metabolism in test experiments.
5
.9.
Catechol 2, 3–dioxygenase enzyme activity
–
1
–1
–1
The activities (U mL ) and the specific activities (mol min mg ) of the C2,3–D enzyme
th
th
are given in Table 5. The activities were determined on the 7 and 14 day of the experiment. 2–
−
1
−1
hydroxymuconic semialdehyde (HMSA) possessed molar extinction coefficient of 36,000 M cm
th
th
[
34]. C2,3–D activity in samples of test experiments on both 7 and 14 day was higher than the
corresponding samples of control experiments. Moreover, specific activity of C2,3D enzyme in
control and test experiments decreased with time. A possible reason underlying this effect could be
inhibition induced by intermediate metabolites of phenanthrene degradation. On relative basis, the
th
reduction in specific activities of the enzymes was higher in control experiments for both 7 and
1
4th day of degradation. This essentially points to relatively lesser inhibition and inactivation of the
cellular enzymes in test experiments. A plausible cause underlying this effect could be enhanced
cellular transport induced by microturbulence generated during sonication. Another possible cause
leading to higher activity of enzymes in test experiments, as demonstrated in previous literature
[35] is alterations in secondary structure of enzyme produced due to sonication. These alterations
include reduction in the rigid –helix content leading to unfolding of proteins that exposes inner
1
9

hydrophobic group and substrate–binding sites. These changes in secondary structure leads to faster
metabolism.
These results clearly show that the enhancement effect of sonication on phenanthrene
biodegradation is mainly of physical nature. The metabolic pathway of degradation is not influenced
by sonication, as phenanthrene degraded through meta– cleavage pathway in both control and test
experiments.
6
.
Conclusion
A mechanistic analysis of sonication–induced enhancement of phenanthrene removal using
C. tropicalis cells has revealed several interesting facets of the bioremediation process. To begin
with, optimum physical parameters for degradation were obtained through the statistical design of
experiments. The enhancement effect of sonication was assessed by replacing mechanical shaking
with sonication (@ 10% duty cycle) during log phase. A 25% rise in phenanthrene degradation was
seen. Analysis of kinetics of degradation revealed strong substrate inhibition for initial
–
1
concentrations > 100 mg L . The biomass yield coefficient increased while the decay coefficient
of the cells reduced in presence of sonication. SDS–PAGE analysis of intracellular protein
expression showed overexpression of total proteins in presence of sonication. Proteins
overexpressed during sonication revealed a band of 35.0 kDa size, which can be predicted as
catechol 2,3–dioxygenase (C2,3–D), an important enzyme involved in phenanthrene degradation
through meta–cleavage route. Activity of C2,3–D enzyme was found to be higher in sonicated
samples, which resulted in higher degradation. A probable cause underlying these effects is
enhanced permeability of cell membranes in presence of sonication that promotes cellular transport
of nutrients, substrate, and metabolites. Finally, flow cytometry analysis showed no adverse effect
of ultrasound on cell viability and morphology. The present study also forms useful basis for further
in–depth investigation on influence of sonication on intracellular proteins and cell metabolism of
Candida tropicalis.
2
0

Acknowledgments
Authors would like to acknowledge infrastructure support provided by Central Instrumentation
facility of I.I.T. Guwahati. Authors also acknowledge Dept. of Biosciences and Bioengineering,
I.I.T. Guwahati for Flow Cytometry analysis.
Conflicts of interest
Authors declare no conflicts of interest of any kind.
Appendix A: Supplementary data
Supplementary data to this article can be found in the online version of the article.
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2
6

Table 1: Summary of literature on polycyclic aromatic hydrocarbon degradation using different microbial strains
Microbial
Strain
PAH degraded
Carbon source
Degradation medium
Initial conc.
Process parameters Degradation Reference
Pichia anomala
Phenanthrene
Phenanthrene
Basal medium (BM)
5.04 mg/L
pH = 5.0-6.0
5.04 mg/L
[9]
Temperature = 27C
Agitation = 150 rpm
Time = 10 days
pH = 5.0-6.0
Temperature = 27C 77.21%
Agitation = 150 rpm 60.77%
Candida
viswanathii
Naphthalene
Phenanthrene
Pyrene
Naphthalene
Phenanthrene
Pyrene
Benzo(a)pyrene + 50g
Tween 80
Basal medium (BM) +
0.5g yeast extract + 10g
glucose
1.79 mg/kg
1.89 mg/kg
1.96 mg/kg
1.98 mg/kg
89.76%
[36]
Benzo(a)pyrene
Time = 10 days
55.53%
Polyporus sp.
S133
Phenanthrene
Phenanthrene +
Tween 80
Mineral salt broth (MSB)
1 mmol/L
50 mg/L
Temperature = 25C 92.00%
Agitation = 120 rpm
Time = 30 days
[37]
[38]
Anthracophyllum
discolor
Anthracene
Phenanthrene
Fluoranthene
Pyrene
Naphthalene
Phenenthrene
Anthracene
Phenanthrene
Fluoranthene
Pyrene
Naphthalene
Phenenthrene
Kirk medium + Tween 80
(0.05% v/v)
pH = 6.6
22.60 %
Temperature = 30C 7.00 %
Time = 28 days
pH = 7.0
19.50 %
8.50 %
97.85%
Candida
tropicalis
Natural salt water (NSW)
+ 0.5% glucose + 0.5%
yeast extract
500 mg/L
25 mg/L
[39]
Temperature = 30C 22.00%
Agitation = 200 rpm
Time = 3 days
Acremonium sp.
P0997
Naphthalene,
Fluorene
Phenanthrene,
Anthracene
Fluoranthene
Phenanthrene
Naphthalene,
Fluorene
Phenanthrene,
Anthracene
Fluoranthene
Phenanthrene
Mineral medium
pH = 6.5
96.90 %
[40]
Temperature = 28C 71.80 %
Agitation = 160 rpm 67.00 %
Time = 15 days
85.00 %
87.90 %
75 %
Pseudomonas sp.
Mineral salt medium
50 mg/L
pH = 7.0
[41]
BZ-3
(MSM) + 20g/L salinity
Temperature = 30C
Agitation = 180 rpm
Time = 7 days
2
7

Table 1 (continued)……
Microbial
PAH degraded
Carbon
Degradation medium
Initial conc.
Process parameters Degradation Reference
Strain
source
Massilia sp. WF1 Phenanthrene
Phenanthrene
Mineral salt medium (MSM)
100 mg/L
pH = 6.0
100 mg/L
80.00 %
98.00 %
[42]
Temperature = 28C
Agitation = 130 rpm
Time = 2 days
pH = 8.0
Temperature = 37C
Agitation = 180 rpm
Time = 30 days
pH = 7.0
Pseudomonas sp.
Phenanthrene
Phenanthrene
Basal medium (BM)
200 mg/L
[2]
W10
Pseudomonas
stutzeri P2
Phenanthrene
Pyrene
Phenanthrene
Pyrene
Carbon-free mineral medium
(CFMM)
1000 mg/L
500 mg/L
[43]
Temperature = 30C 92.60 %
Agitation = 150 rpm
Time = 7 & 10 days
Achromobacter
sp. LH1
Phenanthrene
Phenanthrene
Phenanthrene
Phenanthrene
Mineral salt medium (MSM)
100 mg/L
0.056 mM
Temperature = 30C 94.00%
Agitation = 150 g
Time = 7 days
Temperature = 25C 71.73%
Time = 15 days
[14]
[4]
Trametes hirsuta
D7
Phenanthrene
+ 1% Tween
Mineral salt broth
(MSB)
8
0
Candida
tropicalis MTCC
Phenanthrene Bushnell-Haas (BH) medium 100 mg/L
pH = 6.2
67.00 %
(Present
work)
Temperature =
(Shaking)
84.00 %
*
1
84
33.4C
Agitation = 190 rpm (Sonication)
2
8

Table 2. Factors and levels of central composite experimental design (actual and coded values)
Levels
Factors
Low (–1)
High (+1)
Medium pH (X1)
4
20
130
5
8
40
170
10
Temperature (°C) (X2)
Mechanical shaking (rpm) (X3)
Inoculum size (%, v/v) (X4)
2
9