Bioresource Technology Reports 26 (2024) 101842

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Bioresource Technology Reports

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Interactive effects in biodesulfurization of model oil containing

dibenzothiophene and 4,6-dimethyl dibenzothiophene using Rhodococcus
rhodochrous MTCC 3552
Pushpita Das a, Kuldeep Roy b, Lepakshi Barbora a, Vijayanand Suryakant Moholkar a, c, *
a
School of Energy Science and Engineering, Indian Institute of Technology Guwahati, Guwahati 781 039, Assam, India
b
School of Chemical Engineering, Vellore Institute of Technology (VIT), Vellore 632 014, Tamil Nadu, India
c
Department of Chemical Engineering, Indian Institute of Technology, Guwahati 781 039, Assam, India

A R T I C L E I N F O A B S T R A C T

Keywords: This paper reports investigations into the biodesulfurization process in a dual substrate system. The model system
Biodesulfurization comprises dibenzothiophene (DBT) and 4,6-dimethyl dibenzothiophene (4,6-DMDBT) as substrates and micro­
Dual Substrate bial strains of Rhodococcus rhodochrous. Experiments were conducted for single and dual substrate systems using
Competitive kinetics
different combinations of concentrations. The experimental results were analyzed using mathematical models for
Haldane kinetics
Velocity reduction
competitive kinetics with self-inhibition and competitive kinetics with uncompetitive self- and cross-inhibition.
In the concentration range of 50–150 ppm, the enzyme affinity (Monod constant) of 4,6-DMDBT (4.25–4.83 mM)
is higher than that of DBT (4.83–6.13 mM). As compared to single substrate system, the biodegradation velocity
of DBT reduced to a lesser extent (35–69 %) than 4,6-DMDBT (45–76 %) in dual substrate system. No inhibition
(either self or cross) was seen for the present biodesulfurization system. Retardation of biodegradation velocities
was higher for relatively larger contents of 4,6-DMDBT, plausibly due to a decrease in membrane fluidity and
permeability.

1. Introduction
Removal of heavy aromatic organosulfur compounds such as thio­
phene, benzothiophene, dibenzothiophene, and their derivatives from
liquid fuels (gasoline and diesel) has remained a challenge in petroleum
refining as these compounds have negligible reactivity (recalcitrant)
towards the conventional catalytic hydrodesulfurization. Several mi­
crobial strains belonging to the genera of Rhodococcus, Pseudomonas,
Gordonia, Mycobacterium, Sphingomonas, Paenibacillus, and Arthrobacter
are known to utilize the aromatic organosulfur compounds for their
growth (Das et al., 2020; Nuhu, 2013). Thus, microbial removal of heavy
aromatic organosulfur compounds has emerged as an alternate tech­
nique for deep desulfurization of liquid fuels. Microbial removal of ar­
omatic organosulfur compounds is an eco-friendly process with the
advantage of conserving the fuel's calorific value. Comprehensive re­
views on microbial removal of organosulfur compounds in liquid fuels
have recently been published (Ahmad et al., 2023; Mamuad and Choi,
2023). A critical review of the kinetic modeling of desulfurization using
microbial cultures has been published by Malani et al. (2021) and El-
Gendy and Nassar (2018). More recently, other authors have also pre­
sented kinetic models for sulfur removal using microbial processes
(Nassar et al., 2021; Nassar et al., 2022; Prasoulas et al., 2021; Sadare
and Daramola, 2023). Most previous studies on microbial removal of
organosulfur compounds have used a single substrate system (Abin-
Fuentes et al., 2014; Alcon et al., 2008; Calzada et al., 2012; Martínez
et al., 2017). However, to implement the microbial sulfur removal
process on a large scale, it is necessary to study the facets of this process
in a multisubstrate system.
The biodesulfurization process in a multisubstrate system involves
complex interactions between the substrates. Usually, in multisubstrate
systems, the rate of substrate consumption by the microbial cells is
retarded (compared to individual substrate systems) due to mutual in­
hibition effects between the substrates and toxicity generated by meta­
bolic intermediates/products of these substrates. Depending on the
nature of the substrates (homologous/heterologous), the inhibition
patterns could be competitive or uncompetitive. A few authors (Guch­
hait et al., 2005b; Kobayashi et al., 2001; Zhang et al., 2013) have
studied the inhibitory effects of one substrate on another during

* Corresponding author at: Department of Chemical Engineering, Indian Institute of Technology, Guwahati 781 039, Assam, India.
E-mail address: vmoholkar@iitg.ernet.in (V.S. Moholkar).

https://doi.org/10.1016/j.biteb.2024.101842
Received 14 February 2024; Received in revised form 8 April 2024; Accepted 11 April 2024
Available online 13 April 2024
2589-014X/© 2024 Elsevier Ltd. All rights reserved.
P. Das et al. Bioresource Technology Reports 26 (2024) 101842

Table 1 constants for DBT and DMDBT has given insight into the biomechanism
Kinetic constants in single substrate system. of the simultaneous biodesulfurization of the two organo‑sulfur
(A) Kinetic constants for dibenzothiophene (DBT) compounds.
Initial Kinetic constants
concentrations 2. Materials and methods
Vmax,DBT Ks,DBT Ki,DBT (mM, self-inhibition
(mM⋅h− 1) (mM) constant)
2.1. Chemicals
0.27 mM (50 ppm) 0.472 6.13 9.26
0.54 mM (100 0.602 4.83 18.58
ppm)
Two organosulfur compounds, DBT and 4,6-DMDBT, and model oil,
0.81 mM (150 0.502 5.85 15.24 n-hexadecane, were purchased from Sigma-Aldrich (India). HPLC grade
ppm) n-heptane was procured from S.R.L., India, as a solvent for HPLC. The
other analytical grade chemicals used for the experiments were obtained
from Himedia (India).
(B) Kinetic constants for 4,6-dibenzothiophene (4,6-DMDBT)

Initial Kinetic constants 2.2. Microbial culture maintenance and growth

concentrations
Vmax,DMDBT Ks,DMDBT Ki,DMDBT (mM, self-
(mM⋅h− 1) (mM) inhibition constant)
0.24 mM (50 0.37 4.83 8.21
ppm)
0.47 mM (100 0.43 4.25 15.12
ppm)
0.71 mM (150 0.39 4.43 14.25
ppm)
desulphurization using microbial cultures on a mixture of dibenzothio­
phene (DBT) and its derivatives, viz. 4-methyl dibenzothiophene (4-
MDBT) and 4,6-dimethyl dibenzothiophene (4,6-DMDBT). The mathe­
matical models used in these studies were based on Monod kinetics with
competitive interaction between organosulfur substrates that retarded
the consumption rate of individual organosulfur compounds. A sum­
mary of previous literature on microbial desulfurization in multi­
substrate systems is given in supplementary material (Table S1).
The present study has addressed the issue of gaining insight into
inhibitive interactions among the organo‑sulfur substrates during
simultaneous biodegradation. None of the previous studies have tried to
explore the inhibitory effects and interactions (in terms of self-inhibition
of a substrate and cross-inhibition among multiple substrates) in a
multisubstrate system. The present study has attempted to gain insight
into the inhibitory interactions during biodesulfurization in a multi­
substrate system. More rigorous kinetic models have been developed
that account for the competitive kinetics of two substrates with different
inhibition patterns during degradation of the two organo‑sulfur sub­
strates. These models are based on the hypothesis that the first enzyme
of 4S-pathway (DszC) has multiple binding sites. Thus, the present study
presents a deeper insight into the inhibitory interactions among the
substrates in a practical biodesulfurization system.
The model system used in this study includes two ubiquitous orga­
no‑sulfur compounds found in petroleum fuels, viz. dibenzothiophene
(DBT) and 4,6-dimethyl dibenzothiophene (DMDBT), as substrates. The
microbial strain used to desulfurize the organo‑sulfur substrates is
Rhodococcus rhodochrous MTCC 3552 (or IGST8), which follows the 4S-
pathway. A schematic of the 4S-pathway is provided in the supple­
mentary material (Fig. S1). Previous authors have widely used this wild
strain to effectively desulfurize liquid fuels (Del Olmo et al., 2005;
Gomez et al., 2015; Kaufman et al., 1999; Patel et al., 1997; Prasoulas
et al., 2021). Initially, the individual time profiles of degradation of each
sulfur compound are fitted to the Haldane kinetics model that accounts
for substrate (or self) inhibition. For the dual substrate system, the time
profiles of degradation have been fitted to kinetic models that follow
two inhibition patterns: (1) competitive kinetics with self-inhibition in
which the two substrates compete for the enzyme access, and (2)
competitive kinetics with self-inhibition and uncompetitive cross-
inhibition. The kinetic models have been fitted to the experimental
data using the Genetic Algorithm to yield the numerical values of the
kinetic constants in the model. A comparative analysis of the kinetic
The bacterial strain Rhodococcus rhodochrous MTCC 3552 (ATCC
53968) was procured from the repository of the Institute of Microbial
Technology (IMTECH), Chandigarh, India. The cells were cultivated for
48 h in a sterile Nutrient Broth (NB) medium within an incubator shaker
under controlled conditions, viz. pH = 7, temperature = 30 ◦ C, and an
agitation rate = 200 rpm (Bhasarkar et al., 2015). The process of sub-
culturing was performed every month, and after that, the cells were
maintained at a temperature of − 80 ◦ C in a solution containing 20 % v/v
glycerol.
2.3. Inoculum preparation
Rhodococcus rhodochrous MTCC 3552 cells were cultivated in a
sterilized A2 medium after autoclaving at 121 ◦ C for 20 min. The A2
medium utilized for the experiments had the following composition,
measured in g⋅L− 1: K2HPO4 = 8, KH2PO4 = 6.3, NH4Cl = 2, MgCl2 = 0.2.
Before autoclaving, the medium was supplemented with a metal solu­
tion (2 mL⋅L− 1), and a vitamin solution (1 mL⋅L− 1) was added after
autoclaving (Kirimura et al., 2001). Additionally, a carbon source in the
form of glucose was included at a concentration of 5 g⋅L− 1. The bio­
desulfurization experiments were carried out in an incubator-shaker
(Scigenics Biotech, Model-ORBITEK®) at controlled conditions: pH =
7, temperature = 30 ◦ C, and agitation rate = 200 rpm. The pre-culture
was cultivated for 12 h till reaching the mid-logarithmic growth
phase. In addition, the bacterial biomass was obtained using a centrifuge
followed by rinsing with a phosphate buffer solution. The resultant
biomass pellets, with a dry weight of 0.35 g⋅L− 1, were subsequently
introduced into a sterilized A2 medium.
2.4. Biodesulfurization experiments
2.4.1. Single substrate system
Solutions of DBT and 4,6-DMDBT n-hexadecane were prepared with
the following concentrations: 50, 100, and 150 ppm (or mg⋅L− 1). In
molar units, these concentrations corresponded to 0.27, 0.54, and 0.81
mM for DBT and 0.24, 0.47, and 0.71 mM for 4,6-DMDBT. These con­
centration values were chosen based on previous literature (Liang et al.,
2006; Zhang et al., 2013).
The entire reaction volume was partitioned into seven flasks of 50
mL volume. Each flask contained a total 20 mL solution with organic (n-
hexadecane) to aqueous (A2 medium suspended with cell biomass)
volume ratio of 1:1 (Caro et al., 2007). The pH of the reaction mixture
was maintained at 7. The reaction mixture was incubated at tempera­
ture = 30 ◦ C, agitation speed = 200 rpm. One of these flasks was taken
out after completion of 4, 8, 12, 16, 20, 24, and 28 h of incubation for
analysis. The reaction mixture in the flask was allowed to undergo phase
separation. The organic phase was collected and analyzed for the re­
sidual concentration of the substrate. The experiments were carried out
in duplicate to ascertain the reproducibility of the results.

2
P. Das et al.
Fig. 1. Experimental and simulated biodesulfurization profiles for DBT and 4,6-DMDBT in a single substrate system for different initial concentrations. (A) 50 ppm
(DBT = 0.27 mM, 4,6-DMDBT = 0.24 mM); (B) 100 ppm (DBT = 0.54 mM, 4,6-DMDBT = 0.47 mM); (C) 150 ppm (DBT = 0.81 mM, 4,6-DMDBT = 0.71 mM).
2.4.2. Dual substrate system
The procedure for dual substrate experiments was the same, except
that the organic phase contained both substrates, DBT and 4,6-DMDBT,
in different proportions. The combinations of initial concentrations of
the organosulfur substrates used were as follows: (1) 100 mg⋅L− 1 each of
DBT and 4,6-DMDBT; (2) DBT = 50 mg⋅L− 1, 4,6-DMDBT = 150 mg⋅L− 1;
(3) DBT = 150 mg⋅L− 1, 4,6-DMDBT = 50 mg⋅L− 1. The total initial
concentration of DBT and 4,6-DMDBT was fixed at 200 mg⋅L− 1 for a
plausible comparison of the kinetic constants. In the dual substrate
system, all experiments were repeated twice to assess the reproducibility
of the results.
2.5. Quantification of organosulfur content
Samples of n-hexadecane model oil were collected and filtered
through an Axiva membrane filter with a pore size of 0.2 μm. Normal
phase High-Performance Liquid Chromatography (Agilent Technolo­
gies, 1220 Infinity LC) was used to achieve chromatographic separation
of the organosulfur compounds. A 4.6 × 250 mm NH2 analytical column
(Zorbax) with a 5 μm particle size was used. The flow rate of the mobile
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Bioresource Technology Reports 26 (2024) 101842
phase n-heptane (HPLC grade) was 1 mL⋅min− 1. The mobile phase un­
derwent a prior degassing process through the application of sonication
for 30 min. The diode array detector (DAD) of HPLC was set at a
wavelength of 254 nm.
3. Mathematical model for degradation of substrates
Aromatic organo‑sulfur compounds like DBT and 4,6-DMDBT are
metabolized in the microbial cells of R. rhodochrous occurs via the 4-S
pathway and involves a set of 3 enzymes, viz. DszC, DszA and DszB.
The first two metabolic reactions of the 4S-pathway (for example, DBT
to DBTO and DBTO to DBTO2) are catalyzed by the DszC enzyme, which
is reported to be a tetramer (Gray et al., 1996) and, thus, is likely to have
multiple binding sites. Due to this structural feature, the DszC enzyme is
susceptible to uncompetitive inhibition of self and cross-type. Inhibition
of the DszC enzyme will affect the entire 4S-pathway, affecting the
overall degradation rate of the organo‑sulfur compounds. Given this, the
kinetic models in the present study have been developed using
competitive kinetics with self-inhibition and competitive kinetics with
uncompetitive self- and cross-inhibitions for the DszC enzyme as a
P. Das et al. Bioresource Technology Reports 26 (2024) 101842

Table 2 n is the number of data points in the time profiles of DBT and 4,6-
Kinetic constants in dual substrate system. DMDBT. The root-mean-square error ErrorRMS for fitting the experi­
Combination of initial concentrations Kinetic constantsa,b mental and numerical degradation profiles was defined as:
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
)2̅ √̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
0.27 mM (50 ppm) DBT + 0.71 mM Competitive kinetics with self-inhibition ( exp ( exp )2
(150 ppm) 4,6-DMDBT ErrorRMS = SDBT − Smodel or SDMDBT − Smodel (4)
V′max,DBT = 0.15, V′max,DMDBT = 0.09 DBT DMDBT

Competitive kinetics with Uncompetitive

Sexp exp
DBT and SDMDBT are the experimental concentrations of DBT and 4,6-
self- and cross-inhibition
V″max,DBT = 0.15, K′i,DMDBT = 19.38 DMDBT, while Smodel
DBT and SDMDBT are the concentrations determined using
model

V″max,DMDBT = 0.09, K′i,DBT = 10.25 mathematical model at the same time. The objective function (Obj) was
0.54 mM (100 ppm) DBT + 0.47 mM Competitive kinetics with self-inhibition minimized by manipulating the values of kinetic parameters using a
(100 ppm) 4,6-DMDBT V′max,DBT = 0.20, V′max,DMDBT = 0.15 Genetic Algorithm. This procedure yielded an optimum set of values of
Competitive kinetics with Uncompetitive kinetic parameters for which the numerical and experimental profiles
self- and cross-inhibition matched best, as indicated by the value of regression constant R2
V″max,DBT = 0.21, K′i,DMDBT = 18.13 (Agarwal et al., 2016).
V″max,DMDBT = 0.14, K′i,DBT = 11.16
0.81 mM (150 ppm) DBT + 0.24 mM Competitive kinetics with self-inhibition 3.2. Model for dual substrate system: competitive kinetics with self-
(50 ppm) 4,6-DMDBT V′max,DBT = 0.32, V′max,DMDBT = 0.20
inhibition
Competitive kinetics with Uncompetitive
self- and cross-inhibition
V″max,DBT = 0.31, K′i,DMDBT = 19.77
In a competitive degradation pattern, DBT and DMDBT are simul­
taneously present in the microbial cell and compete independently (with
max,DMDBT = 0.19, Ki,DBT = 17.46
V″ ′
no interactions) to form a complex (ESDBT or ESDMDBT ) with the enzyme,
a
While obtaining the kinetic constants for competitive and uncompetitive which undergoes product formation. However, the enzymatic reaction
kinetics patterns, the values of Monod constants and self-inhibition constants of each substrate is subjected to self-inhibition. The reaction scheme for
have been taken from a single substrate system (Table 1). the biodegradation of DBT in a dual substrate system with competitive
b
The velocities of biodegradation are in mM⋅h− 1 and the inhibition constants
kinetics is shown below:
are in mM.
K s,DBT
representative of the entire 4S-pathway. ESDMDBT ⇄ SDMDBT + E + SDBT ⇄ ESDBT → E + P1
Ks,DMDBT Kcat1
+
3.1. Single substrate system: Haldane kinetic model SDBT
Ki,DBT ↑↓
The Haldane self-inhibition kinetic model is based on the hypothesis ESDBT SDBT
that one substrate molecule forms a primary complex (ESDBT or ESDMDBT ) Different dissociation constants in the reaction scheme are:
with the enzyme, and another substrate molecule binds to the primary
complex to create a secondary non-productive complex (ESDBT SDBT or Ks,DBT =
[E][SDBT ]
⇒[ESDBT ] =
[E][SDBT ]
(5)
ESDMDBT SDMDBT ), which essentially is an uncompetitive self-inhibition [ESDBT ] Ks,DBT
(Palmer and Bonner, 2007). The expressions for biodegradation of
[E][SDMDBT ] [E][SDMDBT ]
DBT and 4,6-DMDBT under self-inhibition mode can be represented Ks,DMDBT = ⇒[ESDMDBT ] = (6)
using the Haldane model for substrates by Eqs. (1) and (2), respectively. [ESDMDBT ] Ks,DMDBT

d[SDBT ] Vmax,DBT [SDBT ]

(1) [ESDBT ][SDBT ] [E][SDBT ]2
− = VDBT = / Ki,DBT = ⇒[ESDBT SDBT ] = (7)
dt Ks,DBT + [SDBT ] + [SDBT ]2 Ki,DBT [ESDBT SDBT ] Ks,DBT Ki,DBT

d[SDMDBT ] Vmax,DMDBT [SDMDBT ] The total enzyme concentration, [ET ], in either free or complexed
− = VDMDBT = / (2) form is:
dt Ks,DMDBT + [SDMDBT ] + [SDMDBT ]2 Ki,DMDBT
[ET ] = [E] + [ESDBT ] + [ESDBT SDBT ] + [ESDMDBT ] (8)
Notations: [SDBT ] and [SDMDBT ] are the concentrations of the DBT and
4,6-DMDBT. VDBT and VDMDBT represent the reaction velocity of desul­ The ratio of enzyme complex of DBT with total enzyme concentra­
furization of DBT and 4,6-DMDBT, respectively. Vmax,DBT and tion is:
Vmax,DMDBT are the maximum reaction velocities (or maximum desulfur­
[ESDBT ] [ESDBT ]
ization rates) for DBT and 4,6-DMDBT, respectively. Ks,DBT and Ki,DBT are = (9)
[ET ] [E] + [ESDBT ] + [ESDBT SDBT ] + [ESDMDBT ]
the Monod (or half saturation) constant and self-inhibition constant for
DBT, whereas Ks,DMDBT and Ki,DMDBT are the Monod (or half saturation) Substitution of Eqs. (5), (6), and (7) in Eq. (9) yields:
constant and self-inhibition constant for 4,6-DMDBT. [E][SDBT ]
The numerical values of the kinetic parameters in these ODEs, viz.: [ESDBT ]
=
Ks,DBT
(10)
Vmax,DBT , Vmax,DMDBT , Ks,DBT , Ks,DMDBT , Ki,DBT , and Ki,DMDBT were obtained
2
[ET ] [E] + Ks,DBT + K[E][S
[E][SDBT ] DBT ]
+ [E][S DMDBT ]
s,DBT Ki,DBT Ks,DMDBT
by fitting experimental time profiles of DBT and 4,6 DMDBT degradation
to Eqs. (1) and (2), which are ordinary differential equations (ODEs) for The reaction velocity for this system is given as: V′DBT = Kcat1 [ESDBT ],
different initial concentrations. Eqs. (1) and (2) were solved in MATLAB and by substituting V′max,DBT = Kcat1 [ET ] and simplifying, we get:
R2022b® using Runge-Kutta 4th order method. To begin with, the nu­
′
merical values of kinetic parameters in these equations were assumed, ′
VDBT =
Vmax,DBT [SDBT ]
( ) (11)
and the numerical solution was compared to the experimental data. The Ks,DBT [SDBT ]2 Ks,DBT
+ [SDBT ] + Ki,DBT + Ks,DMDBT [SDMDBT ]
optimization of the model parameters was done using the objective
function defined as follows:
′
Vmax,DBT [SDBT ]
[ ] d[SDBT ]
∑n − ′
= VDBT = ( ) (12)
Obj = min ErrorRMSi (3) dt
Ks,DBT [SDBT ]2 Ks,DBT
+ [SDBT ] + Ki,DBT + Ks,DMDBT [SDMDBT ]
i=1

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P. Das et al. Bioresource Technology Reports 26 (2024) 101842

Fig. 2. Experimental and simulated substrate degradation profiles in the dual substrate system under competitive kinetics with self-inhibition pattern for different
combinations of initial substrate concentrations. (A) 50 ppm (0.27 mM) DBT + 150 ppm (0.71 mM) 4,6-DMDBT; (B) 100 ppm (0.54 mM) DBT + 100 ppm (0.47 mM)
4,6-DMDBT; (C) 150 ppm (0.81 mM) DBT + 50 ppm (0.24 mM) 4,6-DMDBT.

Analogously, the reaction scheme for enzymatic degradation of

′
DMDBT in a dual substrate system with competitive kinetics is shown −
d[SDMDBT ] ′
= VDMDBT =
Vmax,DMDBT [SDMDBT ]
( )
below: dt
Ks,DMDBT + [SDMDBT ] + [SKDMDBT ]2 K
+ Ks,DMDBT [SDBT ]
i,DMDBT s,DBT

Ks,DMDBT
ESDBT ⇄ SDBT + E + SDMDBT ⇄ ESDMDBT → E + P2 (14)
KsDBT Kcat2
+ The Monod constant (Ks ) and inhibition constant (Ki ) values are
SDMDBT characteristic features of a particular enzyme-substrate system. These
Ki,DMDBT ↑↓ values essentially are a function of the chemical structure of the sub­
ESDMDBT SDMDBT strate and the chemical composition of the enzyme's binding pocket (in
Using the same procedure for DBT, as given previously, the kinetic the present case of DszC enzyme + DBT or DszC enzyme +4,6-DMDBT).
expression for the degradation of DMDBT can be derived as: Thus, these values for a particular substrate are likely to remain the same
even in the presence of other substrates, provided there are no in­
′
Vmax,DMDBT [SDMDBT ] teractions among the substrate molecules (such as the formation of di­
′
VDMDBT = ( ) (13)
[SDMDBT ]2 K mers or temporary bonds) or no significant change in the secondary
Ks,DMDBT + [SDMDBT ] + Ki,DMDBT + Ks,DMDBT [SDBT ]
s,DBT structure of the enzyme in a multisubstrate system. In view of these
conjectures, experimental data was fitted to Eqs. (12) and (14) using the
values of the Monod constants (viz. Ks,DBT and Ks,DMDBT ) and the self-
inhibition constants (viz. Ki,DBT and Ki,DMDBT ) obtained from fitting Eqs.
(1) and (2) to the single substrate data. Previous authors (Kobayashi

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P. Das et al. Bioresource Technology Reports 26 (2024) 101842

Table 3 et al., 2001; Zhang et al., 2013) have also adopted a similar approach for
Reduction in velocities of biodegradation in dual substrate system as compared the kinetic analysis of biodesulfurization in multiple substrate systems.
to single substrate system. V′DBT and V′DMDBT are reaction velocities of desulfurization of DBT and
Combinations of initial Competitive Competitive Reference 4,6-DMDBT, respectively, under competitive kinetics. Maximum reac­
concentrations kinetics with self- kinetics with tion velocities for DBT and 4,6-DMDBT under competitive kinetics are
inhibition Uncompetitive
self- and cross-
denoted as V′max,DBT and V′max,DMDBT , respectively.
inhibition

DBT 4,6- DBT 4,6- 3.3. Model for dual substrate system: competitive kinetics with
DMDBT DMDBT uncompetitive self- and cross-inhibition
0.27 mM (50 ppm) 67.58 76.53 % 69.07 76.53 % This study
DBT + 0.71 mM % % In uncompetitive inhibition kinetics, there is an interaction between
(150 ppm) 4,6- the two substrates during biodegradation. Here, one substrate (DBT or
DMDBT 4,6-DMDBT) binds to the enzyme to form a primary complex (ESDBT or
0.54 mM (100 ppm) 66.61 64.75 % 65.95 67.51 % This study
ESDMDBT ), whereas the second substrate binds to the primary complex to
DBT + 0.47 mM % %
(100 ppm) 4,6- form a non-reactive secondary complex, ESDBT SDMDBT or ESDMDBT SDBT .
DMDBT Thus, the enzyme reaction is inhibited through both self and cross mode.
0.81 mM (150 ppm) 35.66 45.39 % 37.45 47.83 % This study In addition to the uncompetitive cross-inhibition, competitive kinetics
DBT + 0.24 mM (50 % % will always exist as it is a dual substrate system. The reaction scheme for
ppm) 4,6-DMDBT
0.60 mM (110.56 ppm) 24.8 42.8 % – – Zhang
the biodegradation of DBT under uncompetitive inhibition mode is
DBT + 0.37 mM % et al. given as:
(68.18 ppm) 4,6- (2013)
DMDBT

Fig. 3. Experimental and simulated substrate degradation profiles in the dual substrate system under competitive kinetics with uncompetitive self- and cross-
inhibition patterns for different combinations of initial substrate concentrations. (A) 50 ppm (0.27 mM) DBT + 150 ppm (0.71 mM) 4,6-DMDBT; (B) 100 ppm
(0.54 mM) DBT + 100 ppm (0.47 mM) 4,6-DMDBT; (C) 150 ppm (0.81 mM) DBT + 50 ppm (0.24 mM) 4,6-DMDBT.

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P. Das et al. Bioresource Technology Reports 26 (2024) 101842

Table 4
Statistical analysis for the simulations of biodesulfurization in dual substrate system.
(A) Competitive kinetics

Set of experiments Variable Standard error t-Stat p-Value Confidence level (95 %) Student's t-test p value (two-tail)

Lower 95 % Upper 95 %

Set 1 0.27 mM (50 ppm) DBT 0.13 10.63 1.27E− 04 1.05 1.71 0.96
0.71 mM (150 ppm) 4,6-DMDBT 0.01 12.31 6.26E− 05 0.92 1.41 0.93
Set 2 0.54 mM (100 ppm) DBT 0.13 8.92 2.95E− 04 0.80 1.44 0.81
0.47 mM (100 ppm) 4,6-DMDBT 0.16 6.88 9.96E− 04 0.69 1.52 0.91
Set 3 0.81 mM (150 ppm) DBT 0.11 10.94 1.11E− 04 0.91 1.47 0.92
0.24 mM (50 ppm) 4,6-DMDBT 0.11 10.38 1.43E− 04 0.88 1.46 0.99

(B) Uncompetitive inhibition kinetics

Set of experiments Variable Standard error t-Stat p-Value Confidence level (95 %) Student's t-test p value (two-tail)

Lower 95 % Upper 95 %

Set 1 0.27 mM (50 ppm) DBT 0.13 10.65 1.26E− 04 1.06 1.73 0.97
0.71 mM (150 ppm) 4,6-DMDBT 0.09 12.43 5.97E− 05 0.90 1.38 0.86
Set 2 0.54 mM (100 ppm) DBT 0.13 8.57 3.57E− 04 0.76 1.40 0.99
0.47 mM (100 ppm) 4,6-DMDBT 0.17 6.80 1.05E− 03 0.70 1.55 0.93
Set 3 0.81 mM (150 ppm) DBT 0.11 10.87 1.15E− 04 0.91 1.48 0.92
0.24 mM (50 ppm) 4,6-DMDBT 0.12 9.96 1.75E− 04 0.87 1.47 0.92

Set 1 (50 ppm DBT, 150 ppm 4,6-DMDBT), Set 2 (100 ppm DBT, 100 ppm 4,6-DMDBT), Set 3 (150 ppm DBT, 50 ppm 4,6-DMDBT).

ESDBT SDMDBT [ESDBT ] [S ]

↑↓K′i,DMDBT = ( ) DBT (22)
[ET ] Ks,DBT ]2
Ks,DBT + [SDMDBT ] + [SDBT ] + [SKDBT + [SDBT
K′
][SDMDBT ]
SDMDBT Ks,DMDBT i,DBT
i,DMDBT

+
Ks,DMDBT Ks,DBT The reaction velocity for this system is given as: DBT = Kcat1 [ESDBT ], V″
ESDMDBT ⇄ SDMDBT + E + SDBT ⇄ ESDBT → E + P1 and by substituting V″max,DBT = Kcat1 [ET ] and simplifying, we get:
Kcat1
+ ″
Vmax,DBT [SDBT ]
SDBT
″
VDBT = ( ) (23)
Ks,DBT ]2
Ki,DBT ↑↓ Ks,DBT + [SDMDBT ] Ks,DMDBT + [SDBT ] + [SKDBT
i,DBT
+ [SDBT
K′
][SDMDBT ]
i,DMDBT

ESDBT SDBT
d[SDBT ]
Here, ESDBT SDBT is the secondary complex formed through self- − ″
= VDBT
dt
inhibition, while ESDBT SDMDBT is the secondary complex formed ″
Vmax,DBT [SDBT ]
through cross-inhibition. Different dissociation constants in the reaction = ( )
scheme are: Ks,DBT
Ks,DBT
+ [SDMDBT ] Ks,DMDBT + [SDBT ] + [SKDBT ]2
+ [SDBT ][SDMDBT ]
i,DBT K′ i,DMDBT

Ks,DBT =
[E][SDBT ]
⇒[ESDBT ] =
[E][SDBT ]
(15) (24)
[ESDBT ] Ks,DBT
Analogously, the reaction scheme for enzymatic degradation of
[E][SDMDBT ] [E][SDMDBT ] DMDBT in a dual substrate system with uncompetitive kinetics is shown
Ks,DMDBT = ⇒[ESDMDBT ] = (16) below:
[ESDMDBT ] Ks,DMDBT
ESDMDBT SDBT
[ESDBT ][SDBT ] [E][SDBT ]2
Ki,DBT = ⇒[ESDBT SDBT ] = (17) ↑↓K′i,DBT
[ESDBT SDBT ] Ks,DBT Ki,DBT
SDBT
[ESDBT ][SDMDBT ] [ESDBT ][SDMDBT ] +
K′i,DMDBT = ⇒[ESDBT SDMDBT ] = (18) Ks,DBT Ks,DMDBT
[ESDBT SDMDBT ] Ks,DBT K′i,DMDBT ESDBT ⇄ SDBT + E + SDMDBT ⇄ ESDMDBT → E + P2
Kcat2
The total enzyme concentration, [ET ] in either free or complex form, +
is given as: SDMDBT
[ET ] = [E] + [ESDMDBT ] + [ESDBT ] + [ESDBT SDBT ] + [ESDBT SDMDBT ] (19) Ki,DMDBT ↑↓
ESDMDBT SDMDBT
The ratio of enzyme complex of DBT with total enzyme concentra­
tion is: Using the same procedure for DBT, as given previously, the kinetic
expression for the degradation of DMDBT can be derived as:
[ESDBT ] [ESDBT ]
= (20) ″
[ET ] [E] + [ESDMDBT ] + [ESDBT ] + [ESDBT SDBT ] + [ESDBT SDMDBT ] Vmax,DMDBT [SDMDBT ]
″ ( )
VDMDBT =
Substitution of Eqs. (15), (16), (17) and (18) in Eq. (19) gives: Ks,DMDBT
Ks,DMDBT
+ [SDBT ] Ks,DBT + [SDMDBT ] + [SKDMDBT ]2
+ [SDMDBT ][SDBT ]
i,DMDBT K′ i,DBT
[E][SDBT ]
[ESDBT ] (25)
(21)
Ks,DBT
=
[ET ] [E][SDBT ]2
[E] + [E][SDMDBT ] + [E][SDBT ] +
Ks,DMDBT Ks,DBT Ks,DBT Ki,DBT
+ [E][S
K
DBT ][SDMDBT ]
K′
s,DBT i,DMDBT

7
P. Das et al.
d[SDMDBT ] ″ experimental and simulated (after fitting of Eqs. (1) and (2)) profiles of
− = VDMDBT
dt
″
Vmax,DMDBT [SDMDBT ]
= ( ) respectively. It could be inferred from Table 1 that for the same initial
K ]2
Ks,DMDBT + [SDBT ] Ks,DMDBT
s,DBT
+ [SDMDBT ] + [SKDMDBT
i,DMDBT
+ [SDMDBT
K′
The experimental data was fitted to Eqs. (24) and (26), using the
values of the Monod constants (viz. Ks,DBT and Ks,DMDBT ) and the self-
inhibition constants (viz. Ki,DBT and Ki,DMDBT ) obtained from fitting of
Eqs. (1) and (2) to the single substrate data. V″DBT and V″DMDBT are reaction
velocities of desulfurization of DBT and 4,6-DMDBT, respectively, under
competitive kinetics with uncompetitive self- and cross-inhibition mode.
Maximum reaction velocity for DBT and 4,6-DMDBT is denoted as
V″max,DBT and V″max,DMDBT respectively. The cross-inhibition constants of
DBT (for 4,6-DMDBT degradation) and 4,6-DMDBT (for DBT degrada­
tion) under uncompetitive kinetics are represented as K′i,DBT and K′i,DMDBT ,
respectively.
3.4. Fitting of the model to the experimental profiles in dual substrate
system
A similar procedure, as described in Section 3.1 for single substrate
systems, was followed to fit the kinetic model equations (Eqs. (12) &
(14), and (24) & (26)) to the experimental data in the dual substrate
system. Eqs. (12) & (14) and (24) & (26) form two sets of simultaneous
ODEs that were solved using the Runge-Kutta 4th order method (Agar­
wal et al., 2016; Kashyap et al., 2020). The objective function for
obtaining the optimum set of kinetic parameters was defined as follows:
[ ]
∑n
Obj = min ErrorRMSi (27)
i=1
where, n is the number of data points in the time profiles of DBT and 4,6-
DMDBT. The root-mean-square error ErrorRMS for the dual substrate
system was defined as:
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
( exp )2 ( exp
ErrorRMS = SDBT − Smodel + SDMDBT − Smodel
DBT DMDBT
DBT and SDMDBT are the experimental concentrations of DBT and
where Sexp exp
4,6-DMDBT, while Smodel model
DBT and SDMDBT are the concentrations determined
using a mathematical model at the same time.
3.5. Statistical regression analysis
A statistical regression analysis was also performed on the experi­
mental and simulated data to assess the robustness of the model fitting.
The regression analysis provides critical statistical parameters at a 95 %
confidence interval, encompassing t-statistic (or t-stat) value, p-value, F-
value, degrees of freedom (df), correlation coefficient (R2), and standard
error. The outcomes of the F-test and t-test support the assertion that the
model achieves optimal stability under the specified conditions. The
choice of a 5 % significance level aligns with standard practices in sta­
tistical analysis, providing a well-established threshold for determining
the statistical significance of the results. A two-tailed Student's t-test,
assuming homoscedasticity (equal variance), was conducted over the
entire time sequence at a significance level (α) of 5 % (i.e., α = 0.05 or
95 % confidence) (Kashyap et al., 2020; Roy and Moholkar, 2024).
4. Results and discussion
4.1. Analysis of single substrate system
Table 1 and Fig. 1 represent the results of the kinetic analysis of
biodesulfurization in a single substrate system. Fig. 1 shows the
Bioresource Technology Reports 26 (2024) 101842
DBT and 4,6-DMDBT for different initial concentrations. The kinetic
constants for DBT and 4,6-DMDBT are listed in Table 1(A) and (B),
][SDBT ]
concentration (whether 50, 100, or 150 ppm), DBT has higher Ks values
i,DBT
than 4,6-DMDBT. A higher Ks value for DBT means lesser enzyme af­
(26)
finity for ligand binding. However, despite the relatively higher enzyme
affinity for 4,6-DMDBT, the maximum degradation velocity for DBT
(Vmax,DBT ) is faster than that for 4,6-DMDBT (Vmax,DMDBT ). A plausible
explanation for this discrepancy can be given as follows: the degradation
velocity (or rate) of a substrate is a function of two factors, viz. the af­
finity of the substrate towards the enzymes involved in the metabolic
pathway, and secondly, the rate of trans-membrane mass transport of
the substrate molecules (aided by suitable ABC type transporter protein)
(Hirschler et al., 2021). DBT, being a smaller molecule, is expected to
diffuse faster inside the cell, and its population density inside the cell is
expected to be higher than 4,6-DMDBT molecules. This feature essen­
tially results in faster degradation of DBT than 4,6-DMDBT. For all three
initial concentrations of DBT and 4,6-DMDBT, the values of Ki,DBT and
Ki,DMDBT are much higher than the initial concentration, which essen­
tially indicates a negligible self-inhibition effect on substrate
degradation.
For both substrates, Vmax shows a maximum at an initial concentra­
tion of 100 ppm. Rise in Vmax with rise in initial concentration from 50 to
100 ppm could be attributed to the higher availability of substrate inside
the cell. However, as the amount of substrate present in the cell in­
creases, so will be the amounts of intermediates and the end products of
the 4S pathway (viz., 2-hydroxybiphenyl for DBT and 2-(2-hydroxy)3,3′-
dimethylbiphenyl for 4,6-DMDBT). These final metabolic products are
inhibitors of the Dsz enzymes of the 4S pathway (Abin-Fuentes et al.,
2013). Higher accumulation of these products inside the cell leads to
retardation of the metabolic activity. Fall in Vmax for both substrates at
initial concentration of 150 ppm could be attributed to this effect.
Guchhait et al. (2005a) have also analyzed the degradation profiles of
DBT and its alkylated derivatives with an isolated strain of Rhodococcus
sp. (JUBT1) using the Haldane kinetic model. The values of the kinetic
constants with an initial concentration of 100 ppm (0.54 mM) were as
)2 follows: DBT- Ks = 0.54 mM, Ki = 9.01 mM, and 4.6-DMDBT- Ks = 0.78
(28)
mM, Ki = 5.69 mM. The values of the self-inhibition constant obtained
by Guchhait et al. (2005a) are one order of magnitude higher than the
initial concentration, which rules out self-inhibition. Moreover, a
smaller Ks value of 4,6-DMDBT indicates higher substrate affinity than
DBT. Thus, the results of Guchhait et al. (2005a) concur with the present
study.
4.2. Analysis of dual substrate systems
4.2.1. Competitive kinetics with self-inhibition
Table 2 and Fig. 2 represent the results of the kinetic analysis of
biodesulfurization in a dual substrate system with competitive kinetics
and self-inhibition. Fig. 2 shows the experimental and simulated (after
fitting of Eqs. (12) and (14)) profiles of DBT and 4,6-DMDBT for
'
different combinations of initial concentrations. The Vmax values for DBT
and 4,6-DMDBT are listed in Table 2. A comparison of the values of
maximum reaction (or biodegradation) velocities for the same initial
concentrations of DBT and 4,6-DMDBT in single and dual substrate
systems reveals a significant reduction in the velocity of degradation of a
substrate in the presence of another. Percentage reduction in reaction
velocities for DBT and 4,6-DMDBT for different initial concentrations in
competitive kinetics mode are listed in Table 3. A sample calculation for
the velocity reduction is given below.
For initial concentrations of 0.27 mM (50 ppm), DBT has a reaction
velocity of 0.47 mM⋅hr− 1, while for initial concentration of 0.71 mM
(150 ppm), 4,6-DMDBT has a reaction velocity of 0.39 mM⋅hr− 1 (refer to
Table 1). For the concentration combination of 0.27 mM (50 ppm) DBT
8
P. Das et al.
+ 0.71 mM (150 ppm) 4,6-DMDBT, the reaction velocities for DBT and
4,6-DMDBT in case of competitive kinetics with self-inhibition are 0.15
mM⋅hr− 1 and 0.09 mM⋅hr− 1, respectively (refer to Table 2). Thus, the
velocity reduction for DBT is [(0.47 − 0.15)/0.47 ] × 100 = 67.58 %,
while the velocity reduction for 4,6-DMDBT is [(0.39 − 0.09)/0.39 ] ×
100 = 76.53 %. Table 3 reveals higher retardation of 4,6-DMDBT
degradation as compared to DBT in the dual substrate system. This
result is interesting as Ks values of 4,6-DMDBT are smaller than DBT,
indicating a higher affinity towards the enzyme. In competitive kinetics
with self-inhibition, the access to microbial enzymes is divided among
the two substrates, reducing the biodegradation velocities of both sub­
strates. Due to its faster trans-membrane transport, DBT has higher
bioavailability inside the cell, leading to more increased access to mi­
crobial enzymes than 4,6-DMDBT. Thus, the relative retardation
Vmax,DBT is lesser than Vmax,DMDBT .
4.2.2. Competitive kinetics with uncompetitive self- and cross-inhibition
The results of kinetic analysis of biodesulfurization in a dual sub­
strate system with competitive kinetics with uncompetitive self- and
cross-inhibition are given in Table 2 and Fig. 3. The experimental and
simulated (after fitting of Eqs. (24) and (26)) profiles of DBT and 4,6-
DMDBT for different combinations of initial concentrations are shown
in Fig. 3. The values of kinetic constants for DBT and 4,6-DMDBT ob­
tained after fitting the experimental and simulated degradation profiles
are listed in Table 2. For all three combinations of initial concentrations
of DBT and 4,6-DMDBT, the values of cross-inhibition constants for DBT
(K′i,DBT ) and 4,6-DMDBT (K′i,DMDBT ) were found to be much larger than
the initial concentrations, which means there is a negligible cross-
inhibition effect in the biodegradation of both DBT and 4,6-DMDBT.
Nonetheless, for all combinations of initial concentrations of DBT and
4,6-DMDBT, V′max,DBT is higher than V′max,DMDBT . As noted earlier, this
discrepancy can be attributed to faster trans-membrane transport of DBT
molecules, leading to larger bioavailability inside the cell. The reduction
in velocities of DBT and 4,6-DMDBT biodegradation for competitive
kinetics with uncompetitive self- and cross-inhibition are listed in
Table 3. The degradation of 4,6-DMDBT retarded to a greater extent as
compared to DBT for competitive kinetics with uncompetitive self- and
cross-inhibition. Moreover, the extent of velocity reductions in biodeg­
radation of DBT and 4,6-DMDBT in competitive self-inhibition and
competitive kinetics with uncompetitive self- and cross-inhibition sce­
narios are almost similar.
Inspection of the velocity reduction values for different combinations
of initial concentrations of DBT and 4,6-DMDBT reveals that retardation
of biodesulfurization in a dual substrate system is more pronounced in
the presence of higher concentrations of 4,6-DMDBT, which is the larger
organosulfur substrate. For example: (1) Reduction in velocity of 4,6-
DMDBT degradation for the case of 50 ppm DBT + 150 ppm 4,6-
DMDBT in competitive kinetics with uncompetitive self- and cross-
inhibition (76.5 %) is much higher than for the case of 50 ppm 4,6-
DMDBT +150 ppm DBT (47.8 %). (2) The reduction in biodegradation
velocity of DBT for the case 50 ppm DBT + 150 ppm 4,6-DMDBT is 69.1
%, whereas the same for the case 150 ppm DBT + 50 ppm 4,6-DMDBT is
37.5 %. A plausible explanation for these results can be given as follows:
The hydrophobicity of 4,6-DMDBT is higher than DBT due to the pres­
ence of the two methyl groups. Higher hydrophobicity of the substrate
creates a stress response in the lipid composition of the cell membrane of
bacteria (Zumsteg et al., 2023). This effect minimizes the cell membrane
fluidity and permeability, due to which entry of the substrate molecules
in the cells is restricted. This effect is more marked when 4,6-DMDBT is
present in relatively larger quantities. The ultimate manifestation of this
phenomenon is a large reduction of the biodesulfurization velocities of
both 4,6-DMDBT and DBT, as observed experimentally. Zhang et al.
(2013) have also reported reduction in biodegradation velocities of
organo‑sulfur compounds in multisubstrate systems for microbial strain
Mycobacterium sp. ZD-19. For initial concentration combinations of DBT
9
Bioresource Technology Reports 26 (2024) 101842
= 0.64 mM and 4,6-DMDBT = 0.37 mM, the reductions in bio­
desulfurization velocities were determined using Monod kinetics as
24.8 % and 42.8 % for DBT and 4,6-DMDBT, respectively.
4.3. Statistical regression analysis for dual substrate simulations
The statistical analysis assessed the robustness of the kinetic model
by employing various descriptive statistical measures, focusing on the
degradation profiles of dual substrates (DBT and 4,6-DMDBT). The
statistical regression analysis results are presented in Table 4(A) and (B).
Both competitive self-inhibition kinetics and competitive kinetics with
uncompetitive self- and cross-inhibition simulations exhibited statisti­
cally significant differences, with consistent trends in t-stat and p-values
across experimental sets. The two-tailed probability, obtained as p-value
>0.80, was significantly greater than the chosen α level of 0.05, leading
to the rejection of the alternative hypothesis in favour of the null hy­
pothesis. This value suggests no statistically significant difference be­
tween the experimental data and the model predictions.
5. Conclusions
The present study has attempted to gain mechanistic insight into the
biodesulfurization process in a dual substrate system by analysis of
experimental results based on two mathematical models based on
competitive self-inhibition kinetics and competitive kinetics with un­
competitive self- and cross-inhibition. The following conclusions can be
drawn from this analysis:
(1) The biodegradation velocity of DBT and 4,6-DMDBT reduces in a
dual substrate system, where the two substrates compete for ac­
cess to and entry into the microbial cells. Due to faster trans­
membrane transport and higher bioavailability inside the cell,
DBT beats the competition and suffers from relatively smaller
velocity reduction.
(2) The reduction in biodesulfurization velocity of both substrates is
higher when heavier and more hydrophobic 4,6-DMDBT is pre­
sent in larger quantities in the system, which is plausibly a
consequence of the reduction in cell membrane fluidity and
permeability.
(3) The values of self-inhibition (Ki,DBT and Ki,DMDBT ) and cross-
inhibition (K′i,DBT and K′i,DMDBT ) constants are much higher than
the initial concentrations used in this study. Thus, the present
biodesulfurization system does not suffer either self-inhibition or
cross-inhibition.
(4) In a single substrate system, reduction in biodegradation velocity
occurs for DBT and 4,6-DMDBT at relatively higher substrate
concentration – plausibly due to inhibition of Dsz enzymes by
metabolites and final products of the 4S-pathway.
Further investigations in biodesulfurization in multisubstrate can be
carried out along the following lines: (1) the kinetic models reported in
this study can be further developed for multisubstrate systems for other
organosulfur compounds, (2) determination of the profiles of in­
termediates and final product of 4S-pathway, (3) determination of the
total sulfur removal from the organic phase.
Funding
This work was financially supported by the Council of Scientific and
Industrial Research (CSIR), India, through extramural grant no. 22/
0806/19/EMR-II.
CRediT authorship contribution statement
Pushpita Das: Writing – original draft, Methodology, Investigation,
P. Das et al.
Formal analysis, Conceptualization. Kuldeep Roy: Software, Investi­
gation, Formal analysis. Lepakshi Barbora: Supervision, Project
administration. Vijayanand Suryakant Moholkar: Writing – review &
editing, Supervision, Conceptualization.
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.
Data availability
No data was used for the research described in the article.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biteb.2024.101842.
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