Journal of Petroleum Science and Engineering 160 (2018) 412–421

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Journal of Petroleum Science and Engineering

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

Kinetic and thermodynamic behavior of the biodegradation of waxy crude

oil using Bacillus subtilis
N. Sakthipriya a, Mukesh Doble b, Jitendra S. Sangwai a, *
a
Flow Assurance Laboratory, Petroleum Engineering Program, Department of Ocean Engineering, Indian Institute of Technology Madras, Chennai 600 036, India
b
Bio Engineering Laboratory, Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600 036, India

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

Keywords: The present work investigates the reaction kinetics of biodegradation of waxy crude oil using Bacillus subtilis YB7,
Biodegradation a thermophilic organism. Mathematical modeling of the reaction kinetics of microbial growth together with
Biosurfactant biosurfactant production and hydrocarbon substrate utilization have been attempted. Although various models,
Kinetic modeling such as Monod's, Logistic, Tessier and Contois were investigated for substrate degradation and biomass produc-
Monod's model tion, and compared with the experimental studies, none of them could accurately predict the biosurfactant
Thermodynamics
production in the presence of waxy crude oil. In this study, a new model, which is an extension of Monod's
equation, has been developed to predict the observed values of biosurfactant production along with substrate
degradation and biomass production in the presence of waxy crude oil satisfactorily. The proposed model has also
been tested with various experimental studies carried out in the literature. The proposed model is observed to
provide satisfactory model predictions when compared to other models available in literature. This shows that the
proposed model can be used to study the reaction kinetics of all types of biodegradation studies to find the op-
timum parameters for maximum possible biodegradation of hydrocarbon substrates and production of bio-
surfactant. The positive values of activation energy observed in the study indicates that the process of
biodegradation is an endothermic process. The negative values of entropy indicates that the reacting molecule
underwent an internal rearrangement to give the activated complex, without a change in the number of mole-
cules. It is believed that this study will be suitable for possible model development for biodegradation of hy-
drocarbons for oil recovery applications or bioremediation methods.

1. Introduction recovery and oilfield operations. Kinetic studies provide a theoretical

Biodegradation of petroleum hydrocarbons has received an
increasing awareness in the recent years owing to its potential as a
platform for the bioremediation, enhanced oil recovery and other
onshore and offshore oil spill remedial issues. Studies involving micro-
bial growth, effect of nutrients on growth kinetics, and their impact on
biodegradation of hydrocarbon forms an integral part of the whole pro-
cess. Many studies in the literature were focused on microbial enhance-
ments and manipulation of nutrient medium, and control feeding to
improve the degradation of hydrocarbons (Noordman and Janssen, 2002;
Etoumi, 2007; Bao et al., 2014; Tehrani and Herfatmanesh, 2015;
Ahmadi et al., 2017). Few studies have addressed the influence of sub-
strate in the fermentation and their kinetics (Schmidt et al., 1985; Choi
et al., 1999). However, information on the kinetic models of biodegra-
dation of hydrocarbons need improvement, particularly for enhanced oil
framework for optimal design of the process utilizing microbial tech-
nique for process development (Sivakumar et al., 1994).
Several authors reported various kinetic models representing micro-
bial growth and substrate utilization in biological methods (Kumar et al.,
2005; Nakhla et al., 2006; Lin et al., 2008). The kinetic parameters were
determined using linear form of kinetic models. Mainly two approaches,
such as non-inhibitory and inhibitory have been presented in the litera-
ture for representing the kinetics of microbial growth of single substrate.
Monod (1949) has successfully developed a simplest non-inhibitory ki-
netic relation between microbial growth and substrate concentration.
Monod's equation is applied to the degradation of substrates at concen-
tration which saturate the activity of microbial cells partially. Several
studies have been accomplished on the microbial growth and kinetics of
degradation of hydrocarbon substrate using the Monod's model (Prats
and Rodriguez, 1992; Nakhla et al., 2006; Ostendorf et al., 2007; de

* Corresponding author.
E-mail address: jitendrasangwai@iitm.ac.in (J.S. Sangwai).

https://doi.org/10.1016/j.petrol.2017.10.056
Received 9 June 2017; Received in revised form 4 October 2017; Accepted 19 October 2017
Available online 23 October 2017
0920-4105/© 2017 Elsevier B.V. All rights reserved.
N. Sakthipriya et al. Journal of Petroleum Science and Engineering 160 (2018) 412–421

Table 1
Various kinetic models used in this study.a

Model μ Parameters Reference

μmax S μmax, KS, YX/S and
Monod's μ¼ Ks þS
Monod, 1949
model YP/X
Tessier μ ¼ μmax ð1  expðS=TÞÞ μmax, T, KS, YX/S and Tessier, 1936
model YP/X
Contois μ¼ μmax S μmax, B, KS, YX/S and Contois, 1959
BXþS
model YP/X
 
Logistic μmax, Xmax, YX/S and Kumar et al.,
μ ¼ μmax 1  Xmax
X
model YP/X 2005
Present μ ¼ μKmax S
 BX μmax, B, KS, YX/S and
s þS S
work YP/X
a
Xmax - maximum biomass concentration, g L1; μ - specific growth rate of microor-
ganisms, h1; μmax - maximum growth rate, h1; B - coefficient of biomass; and KS - sub-
Fig. 3. Characteristics of crude oil degradation using Bacillus subtilis at 50  C.
strate saturation constant, g L1.

Fig. 4. Growth kinetics of Bacillus subtilis at various temperature.

Fig. 1. Growth of Bacillus subtilis on waxy crude oil as a function of temperature.

concentration and microbial population. Most of the models, such as

Oliveira et al., 2013; Sakthipriya et al., 2015a). Other than Monod's
model, many other models are being used for the description of
biodegradation kinetics. Simkins and Alexander (1984) developed a
non-linear and non-inhibitory kinetic model for aqueous systems. He
observed that the growth rate and saturation constant are valuable means
for analysis of microbial kinetics. Contois (1959) developed a model in
which the half saturation rate is presumed to be dependent on the
biomass concentration. Tessier (1936) has developed a model based on
an exponential function.
Logistic model approximates the Monod equation by integration at the
substrate concentration below the half saturation constant for growth of
the active cells (Robinson and Tiedje, 1983; Simkins and Alexander, 1984).
Mercier et al. (1992) developed a model based on the logistic model
describing biomass growth, substrate utilization and product formation.
All the above mentioned kinetic models depend on initial substrate
Fig. 2. GC-MS image of waxy crude oil before degradation and after degradation with
various temperatures.
Contois, Micheles-Menton, Andrews, etc., in the literature are acceptable
extensions of the Monod model except the Tessier model (Contois, 1959;
Tessier, 1936; Prats and Rodriguez, 1992). Another approach in the mi-
crobial kinetics considers the substrate as inhibitory compound and many
studies and models have been developed for substrate inhibition studies.
Inhibitory kinetics is performed for the reaction where the enzymes are
inhibited by their own carbon substrate and is considered as an annoyance
for the biochemical reaction (Reed et al., 2010). Lin et al. (2008) devel-
oped the substrate inhibition kinetic model for the fermentation using
Actinobacillus succinogenes and observed its optimum ability to grow in
glucose substrate. All of the above mentioned studies were focused on the
kinetics of the growth of microorganisms or the consumption of environ-
mental contaminants (de Oliveira et al., 2013). It is important to correlate
the relation between the crude oil degradation and the amount of bio-
surfactant produced. Biosurfactant plays significant role in the degradation
of crude oil, hence, it is necessary to find the relationship between them.
The kinetics of biosurfactant production by microorganisms will be of a
great interest to scientists to scale-up the process.
In our previous study, we have validated the biosurfactant production
along with paraffin degradation, and biomass production using Pseudo-
monas aeruginosa and Pseudomonas fluorescens using Monod and Logistic
model (Sakthipriya et al., 2015a). In that study (Sakthipriya et al.,
2015a), we have observed that the Monod's model was successful in
predicting the performance of the process only at the initial growth
period, while the logistic model has shown the regression coefficient of
0.98. An improved kinetic model which can fits the experimental values
on biosurfactant production, substrate degradation, and microbial
growth is necessary. Hence, in this study, we have intended to develop a
new model by incorporating certain parameters in already existing
Monod's model to encompass biomass, biosurfactant production and
carbon substrate utilization.

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N. Sakthipriya et al. Journal of Petroleum Science and Engineering 160 (2018) 412–421

Table 2
Comparison of microbial growth rate with degradation of waxy crude oil and production of surfactin at 48 h of incubation.a

Temperature,  C μ td, R2 S P,

35 0.075 ± 0 .0016 9.25 ± 0.12 0.96 0.41 ± 0.23 1.02 ± 0.08

50 0.084 ± 0.013 8.25 ± 0.1 0.99 0.31 ± 0.17 1.19 ± 0.21
75 0.071 ± 0.005 9.76 ± 0.20 0.98 0.45 ± 0.09 0.93 ± 0.01
a
μ-Specific growth rate of microorganisms, h1; td-Doubling time, h; S-Initial rate of the substrate consumption, g L1d1; P-Initial rate of biosurfactant produced, g L1d1; Results are
represented with mean ± standard deviation of three independent experiments.

The current study involves the mathematical modeling of the reaction
kinetics incorporating microbial growth, biosurfactant production and
substrate utilization using Bacillus subtilis YB7 a thermophilic organism.
Formulation of an appropriate mathematical model serves as a guide to
microbial process developments with relevance to degradation. Various
models have also been compared with the experimental results and the
new model has been developed to get the best fit with the observed
experimental values. The developed model has been compared with the
experimental values on biosurfactant production, substrate degradation,
and microbial growth taken from our previous studies (Sakthipriya et al.,
2015b, 2015c) and other data available in literature. Other than kinetic
studies, thermodynamic behavior of biodegradation is necessary to check
the feasibility of the process for large scale industrial applications. Such
studies are not common in the literature dealing with the biodegradation
of petroleum fractions. Hence, in this study, thermodynamic behavior of
biodegradation of hydrocarbon has also been investigated.
2. Biodegradation experiments
hydrocarbon is relative to the total area of peaks. The percentage
degradation of waxy crude oil was evaluated by matching the area of
the peaks acquired before and after the crude oil degradation in the
chromatograph as obtained from GC-MS. The collected samples were
also analyzed for the production of biosurfactant (Cooper et al., 1981),
microbial count (colony forming units) (Hassanshahian et al., 2014),
and biomass dry weight (Etoumi, 2007). All the experiments were
carried out in triplicates and the mean values were reported in
Figures and Tables. Error bars in the figures represent the standard
deviation of triplicates.
3. Reaction kinetics
The specific growth rate (μ, h1) of the microorganism is calculated
from Eq. (1).
dX
¼ rx ¼ μX (1)
dt

where, X is biomass dry weight at time t (h), g L1; rx is biomass pro-

Experiments on biodegradation of waxy crude oil has been presented
duction rate, g L1 h1; μ is calculated by integrating Eq. (1) with con-
here in a brief. Details of the experiments and procedure for the culti-
ditions, X ¼ Xo at t ¼ to (Kumar et al., 2005),
vation of Bacillus subtilis on waxy crude oil substrate have been presented
in our previous study (Sakthipriya et al., 2015b). The biodegradation of ln X  ln Xo
waxy crude oil using Bacillus subtilis YB7 (GenBank accession number: μ¼ (2)
t  to
GQ241354) was conducted in 250 mL conical flask containing 100 mL of
nutrient medium and 2% of waxy crude oil as a sole carbon and energy μ is determined by plotting ln (X/Xo) vs. time. Doubling time (td), a time
source. Waxy crude oil was obtained from Mehsana oilfield asset, requisite for the microorganism to double from its initial count, is
Gujarat, India. The waxy crude oil contains 67.75 ± 3.40% saturates, calculated as,
22.66 ± 1.10% aromatics, 8.87 ± 0.45% resins and 0.72 ± 0.04%
asphaltenes. The nutrient medium contains 1.5 g L1 of potassium ln 2
td ¼ (3)
dihydrogen phosphate, 3 g L1 of disodium hydrogen phosphate, 1 g L1 μ
of ammonium chloride, 2.5 g L1 of sodium chloride, 4 g L1 of glucose,
and 0.05 g L1 of magnesium sulfate hepta hydrate in 200 mL of distilled
water. Flask without crude oil was used as a control. Experiments were
carried out at 35, 50 and 75  C in a shaker (Orbitek, Scigenics Biotech, 3.1. Decay coefficient
India) at 180 rpm.
In order to analyze biodegradation of waxy crude oil under the in- Typically, a growth curve of microorganism shows a drop in microbial
cubation conditions, the concentration of waxy crude oil has been population after complete utilization of the substrate. To determine the
determined at different time intervals. The concentrated hydrocarbon decay coefficient, the incubation of microorganism was not stopped, and
extract from the culture supernatant (after discarding the biomass) was
examined using gas chromatography-mass spectroscopy (GC-MS) (GC-
Mate II, JEOL, USA). It has been established that the concentration of

Table 3
First order rate constant of biodegradation of waxy crude oil using Bacillus subtilis.

Degradation period Temperature, K k, s1

Day 1 308 1.1  105

323 1.4  105
348 9.2E-06
Day 5 308 3.0  106
323 5.1  106
348 2.6  106
Day 10 308 2.2  106
323 3.8  106
348 1.9  106
Day 15 308 1.6  106
323 2.7  106
Fig. 5. Evaluation of decay coefficient k for Bacillus subtilis on waxy crude oil using decay
348 1.3  106
phase batch growth experimental data.

414
N. Sakthipriya et al. Journal of Petroleum Science and Engineering 160 (2018) 412–421

Fig. 6. Plot to calculate yield coefficients for (a) biosurfactant produced per waxy crude oil utilized; (b) biomass produced per waxy crude oil utilized; (c) biosurfactant produced per
biomass produced.

the measurement of optical density (OD) using UV spectrophotometer (V P  P0

YP=S ¼ (8)
550, Jasco, US) was continued even after the complete utilization of S0  S
waxy crude oil. During this dead phase, some of the cells become the
carbon and energy source for other cells. This dead phase of the growth P  P0
curve in a batch scale reactor is determined by (Kumar et al., 2005). YP=X ¼ (9)
X  X0
dX
¼ kd dX (4) where, YP/X is the yield of the biosurfactant per cell weight, g g1; YP/S is
dt the yield of the biosurfactant per substrate utilized, g g1; P0 indicates the
biosurfactant concentration at initial time, g L1.
where, kd is the decay coefficient, h-1; Slope of the graph obtained by
plotting ln (OD) versus time gives kd.
3.3. Kinetic models

3.2. Yield coefficient The experimental values on biomass dry weight, biosurfactant pro-
duction and the amount of waxy crude oil utilization has been compared
The quantity of the biomass existing at the end of exponential growth using various kinetic models reported in the literature, such as the
phase was used to estimate the yield coefficient of biomass (YX/S) in the Monod, Tessier, Contois and logistic (Tessier, 1936; Monod, 1949;
presence of hydrocarbons. YX/S can be calculated from the Contois, 1959; Kumar et al., 2005). In addition to these models, a new
following equation. model has been developed in this work with the help of basic Monod's
model incorporating the biomass production. According to Lee (2006),
dS 1 dX
¼ (5) the microbial growth rate is directly proportional to the biomass dry
dt YX=S dt
weight during the exponential growth phase under standard nutrient
Integrating Eq. (5) with limits S0 to S, and X0 to X, gives, condition. Hence, we have incorporated biomass dry weight ‘X’ in our
model. Table 1 gives the information on various models and their pa-
X  X0 rameters used in this study. The best fit of the model was identified with
YX=S ¼ (6)
S0  S the help of regression coefficient (R2). The R2 value suggests the most
suitable kinetic model and the kinetic parameters associated with
where, X0 is initial biomass concentration, (g L1); S and S0 are substrate that model.
(crude oil) concentration at any time and initial substrate concentration, The specific growth rate in Eq. (1), was replaced by different forms
(g L1). YX/S is the yield of biomass per substrate utilization, (g g1). The as given in Table 1. Table 1 also provides the equation for specific
yield coefficient for biosurfactant production (P) and crude oil utilization growth rate suggested in this work as an extension to the Monod's
(S) can be calculated according to the relationship reported by model. The modified set of equations were solved using MatLab® 2010
Hong (1989). (The math works, USA) with the help of a function ode45. The kinetic
parameters, such as μmax, KS, B, f, YX/S and YP/X were estimated by
dP dX
¼ YP=X (7) assuming the initial set of parameter values. Generally, the initial
dt dt values will be obtained from the experimental results. The sum of
Combining Eqs. (1), (5) and (7), we get: squares of the error between the predicted values and the experimental

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N. Sakthipriya et al. Journal of Petroleum Science and Engineering 160 (2018) 412–421

5 4

4
3

X,S and P (g/L)

X,S and P (g/L)
2
2
1
1
(a) (b)
0
0

-1 -1
0 200 400 600 800 0 200 400 600 800
Time (h) Time (h)
4 4

3
3

X,S and P (g/L)

X,S and P (g/L)

2
2
1

1
(c) 0 (d)

0 -1
0 200 400 600 800 0 200 400 600 800
Time (h) Time (h)
4

3.5

3
X,S and P (g/L)

2.5

1.5

1
(e)
0.5

0
0 200 400 600 800
Time (h)

Fig. 7. Kinetic modeling, (a) Monod's model; (b) Contois model; (c) Tessier model; (d) Logistic model; (e) Present model; Circles-Experimental values; Red color-Biosurfactant production
(P); Blue color-Biomass dry weight (X); Green color-Waxy crude oil degraded (S). (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)

(R2) was determined. The set of values giving the highest R2 was pre-
dicted. In all the models, the parameters μmax, KS, YP/X and YX/S were
taken from the experimental outcomes for preliminary guess and tuned
to determine a satisfactory match between the experimental and pre-
dicted data.
4. Thermodynamic studies
Thermodynamic parameters, namely, enthalpy (H), entropy (S) and
Gibb's free energy (G) are important to estimate the feasibility of the
biodegradation process. The entropy of activation is calculated as (Low
et al., 1973),

Table 4
Model parameters for the rhamnolipid formation, substrate utilization and biomass growth.

Model KS A YX/S R2 YP/X R2 μmax R2 Xmax

Monod's model 1.2 – 2.26 0.79 1.96 0.20 0.1 0.97 –

Logistic model – – 2.2 0.80 1.67 0.84 9.064 0.98 2.58
Contois model 1 18.8 2.19 0.59 1.68 0.67 0.64 0.81 –
Tessier model 1.6 – 2.56 0.95 1.06 0.78 0.34 0.76 –
Present model 4.95 1 2.3 0.99 1.65 0.95 0.76 0.99 –

*KS-Saturation constant, g L1; A-factor depending on biomass dry weight; μmax -Maximum growth rate of microorganism, h1; YP/X-Yield of the rhamnolipid on cell weight,g g1; YP/S- Yield
of rhamnolipid on substrate, g g1; YX/S- Yield of biomass on substrate, g g1.

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N. Sakthipriya et al. Journal of Petroleum Science and Engineering 160 (2018) 412–421

kB T obtained by plotting ln k versus 1/T. The value of the slope is equal to

k¼  expðΔS=RÞ (10)
h -Ea/R. A is calculated from the intercept of the graph. The Gibbs free
energy of activation (ΔG), J mol1 can be calculated as,
where, k is first order rate constant (s1); kB is Boltzmann's constant (J
K1); h is Planck's constant (Js); T is temperature (K); ΔS is entropy of ΔG ¼ ΔH  TΔS (14)
activation, (J K1 mol1) and R is the universal gas constant (J
K1mol1). Enthalpy of activation is calculated as, 5. Results and discussion

ΔH ¼ Ea  RT (11) 5.1. Characteristics of Bacillus subtilis

where, Ea is activation energy (J mol1) and ΔH is enthalpy of activation

The growth of the microorganisms is a very complex phenomenon
(J K1); The activation energy, which is necessary to initiate the
(Sivakumar et al., 1994). Many scientists have performed the incubation
biodegradation was calculated using the Arrhenius equation as
of Bacillus subtilis at 37 to 42  C (Ghojavand et al., 2012; Vaz et al., 2012;
shown below.
Al-Wahaibi et al., 2014). Though Bacillus subtilis is thermophilic in na-
Ea ture, earlier it was not utilized at high temperature crude oil degradation.
k ¼ AeRT (12)
However, recently, in our studies, we have utilized Bacillus subtilis for the
Taking logarithm of Eq. (2), we get, degradation of crude oil at 75  C (Sakthipriya et al., 2015b). Fig. 1 dis-
plays the growth of Bacillus subtilis on waxy crude oil substrate at various
Ea temperatures. Maximum concentration of Bacillus subtilis on the crude oil
ln k ¼ ln A  (13)
RT was observed to be 28, 32, and 28  106 CFU/mL at 35, 50, and 75  C,
respectively (Sakthipriya et al., 2015b). The concentration of
where, A is pre-exponential factor, s1; Ea was calculated from the slope

3.5 3.5

3
(a)
3

2.5 2.5 (b)

S and P (g/L)
S and P (g/L)

2 2

1.5 1.5

1 1

0.5 0.5

0 0
0 10 20 30 40 50 60 0 20 40 60
Time (h) Time (h)

(c)
2.5

2
S and P (g/L)

1.5

0.5

0
0 10 20 30 40 50 60
Time (h)

Fig. 8. Model validation of substrate utilized and biosurfactant produced; (a) Present model; (b) Monod's model; (c) Tessier model; Circles-Experimental values of Nitschke and Pastore
(2006); Red color-Biosurfactant production (P); Blue color-Waxy crude oil degraded (S). (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)

417
N. Sakthipriya et al. Journal of Petroleum Science and Engineering 160 (2018) 412–421

microorganisms has increased upto 10 days, and decreased thereafter.
The maximum growth of bacteria was found to be at 50  C rather than at
35 and 75  C. An increase in the number of active cells increases the
degradation rate. This indicate the efficacy of the Bacillus subtilis YB7 for
effectual biodegradation of hydrocarbons appropriate for oil recovery
from reservoirs, bioremediation, and flow assurance studies.
Bacillus subtilis is observed to produce a biosurfactant called surfactin.
Various researchers have observed that the Bacillus subtilis can produce
surfactin in the range of 0.025–3 g L1 at 28 to 40  C (Grishchenkov et al.,
2000; Nitschke and Pastore, 2006; Salehizadeh and Mohammadizad,
2009; Darvishi et al., 2011; Al-Bahry et al., 2013). We also have reported
similar studies on the production of surfactin in the presence of crude oil
as used in this investigations. Details of the biosurfactant experiments
used for validation here is presented elsewhere (Sakthipriya et al., 2015b,
2015c). The adequacy of the biosurfactant production and the detailed
measurements were given in our previous reported study (Sakthipriya
et al., 2015c) and are not reproduced here for the sake of brevity. In the
previous study, we have analyzed the production and characterization of
surfactin using various hydrocarbon substrates at various temperatures
elaborately and observed maximum of 4 g L1 of surfactin at 50  C
(Sakthipriya et al., 2015c).
The biodegraded samples of crude oil were fractionated using GC-MS,
and the extent of biodegradation was determined (Sakthipriya et al.,
2015b.). The effect of temperature on the rate of biodegradation of has
been studied by incubating microorganism with crude oil at various
temperatures, such as 35, 50 and 75  C. Fig. 2 shows the chromatograms
of waxy crude oil before degradation and after degradation at 35, 50 and
75  C. Comparing the area under the peaks of chromatographs before and
after degradation, it has been observed that the maximum 96% of waxy
crude oil was degraded in 10 days, and negligible degradation was
observed thereafter. This result is in good agreement with the microbial
growth (see Fig. 1). The crude oil degradation was 80 and 85% after 10
days of incubation at 75 and 35  C, respectively. This indicates that the
incubation temperature has pronounced impact on the degradation of
hydrocarbons under biodegradation conditions. Fig. 3 shows the char-
acteristic of Bacillus subtilis at the optimum temperature of 50  C. It is
clearly understood that production of biosurfactant directly impacts
crude oil degradation.
5.2. Reaction kinetics
Fig. 4 shows the plot for growth kinetics of Bacillus subtilis at various
temperature with waxy crude oil as a carbon source. The graph was
plotted for ln (X/Xo) versus incubation time. The slope of the plot gives
the specific growth rate of the microorganism (Eq. (2)). The comparison
of microbial growth rate with degradation of paraffin and production of

0.5 0.5

0.4 (a) 0.4

(b)

0.3 0.3
X(g/L)

X(g/L)

0.2 0.2

0.1 0.1

0 0
0 50 100 150 200 250 0 50 100 150 200 250
Time (h) Time (h)

0.4
(c)

0.3
X (g/L)

0.2

0.1

0
0 50 100 150 200 250
Time (h)
Fig. 9. Model validation of biomass produced; (a) Present model; (b) Monod's model; (c) Tessier model; Circles-Experimental values of Zhang et al. (2005).

418
N. Sakthipriya et al.
surfactin is given in Table 2. The specific growth rate was observed to be
0.075 ± 0 .0016, 0.084 ± 0.013 and 0.0710.071 ± 0.005 h1, respec-
tively, at 35, 50 and 75  C. The corresponding values of doubling time are
9.25 ± 0.12, 8.25 ± 0.1, and 9.76 ± 0.20 h. The doubling time at 50  C is
found to be lesser than that incubated at 30 and 75  C.
The concentrations of waxy crude oil degraded (S) has been deter-
mined by gas chromatographic techniques at various time intervals.
Initially, the reaction is assumed to follow the first order kinetics and the
graph was plotted for ln(S/So) against incubation time and it gave
straight lines. The R2 values were found to be 0.9 for all the incubation
temperatures, which suggests that the biodegradation of waxy crude oil
follows the first order reaction. The first order rate constants were
calculated from the slopes of these lines and are given in Table 3.
Fig. 5 shows the growth curve of Bacillus subtilis at 50  C extended
upto the endogenous region. The values of natural logarithm of optical
density was plotted against incubation time and the value obtained
from the slope of the graph gives the decay coefficient. According to
Kumar et al. (2005), the value of kd is independent of the initial sub-
strate concentration. The value of the coefficient of decay rate was
observed to be 0.00003 h1 for waxy crude oil. Decay coefficient
directly affects the growth rate. Due to this, the wash out condition
(growth cannot be sustained) will occur at low dilution rate. This will
be very important in case of continuous flow mixed reactors during
large scale applications.
Fig. 6 shows the plots to determine yield coefficients for biosurfactant
produced per waxy crude oil utilized (YP/S); biomass produced per waxy
crude oil utilized (YX/S) and biosurfactant produced per biomass pro-
duced (YP/X), respectively. The amount of biomass available immediately
at the end of exponential growth phase was used in estimating the
biomass produced as a result of consumption of substrate. According to
Pirt (1975), the observed yield coefficient is calculated as,
1 1 m
¼ þ (15)
Y YT μ
where, Y is the observed yield coefficient, YT is the true yield coefficient
and m is the maintenance coefficient. In case of batch system, the growth
rate ‘μ’ does not vary significantly from μmax. Hence, any discrepancy
arises because of the effect of maintenance requirement on the substrate
utilization is neglected (Kumar et al., 2005). Thus, the observed yield
coefficient is equal to the true yield coefficient. This observation is also
supported by the negligible decay ratio. The value of YX/S in the presence
of waxy crude oil was observed to be 1.99 g/g, with the regression co-
efficient (R2) of 0.98. The values of YP/X and YP/S were observed to be
1.68 and 3.36 g/g, respectively, and the corresponding R2 values were
observed to be 0.99 and 0.96.
5.3. Correlation of kinetics models
Fig. 7 shows the comparison between the experimental data with the
prediction of various kinetic models. Monod's model which forms the
foundation for many models matches only with biomass production
(R2 ¼ 0.97) and substrate degradation (R2 ¼ 0.79). Monod's equation has
fitted the experimental values of biosurfactant production only during
the initial degradation time (but not with the whole reaction time,
R2 ¼ 0.20). This observation is in-line with Lin et al. (2000), who stated
that the Monod model is applicable only during the exponential growth
phase (Fig. 7a). Contois model (see Fig. 7b) fits the observed values in all
the three plots of X, P and S with R2 of 0.81, 0.67 and 0.59, respectively.
The corresponding R2 values using Tessier model (Fig. 7c) are 0.76, 0.78
and 0.95. The values of the model parameters for the biosurfactant
production, substrate (crude oil) utilization and biomass growth are
given in Table 4. Logistic model developed by Mercier et al. (1992)
matches the X and S with the correlation coefficient of 0.98 and 0.80 (see
Fig. 7d). This model matches the biosurfactant production with R2 of
0.84. All the models described above did not fit all X, S, and P perfectly. If
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Journal of Petroleum Science and Engineering 160 (2018) 412–421
the model fits X and S, it did not satisfy P. The model values fitting the
experimental results of stationary growth phase did not match with
observed values during exponential growth phase. Hence, in order to
overcome this difficulty, we have proposed a model incorporating the
term ‘X’ in the basic Monod's equation (Table 1). The proposed model
gives the best fit for biomass production (R2 ¼ 0.99) and substrate uti-
lization (R2 ¼ 0.99). The proposed model has also fitted the biosuractant
production perfectly (R2 ¼ 0.95) compared to any other model in the
literature (Fig. 7e).
The proposed model has also been compared with other experiment
values reported in the literature. The values of biosurfactant produced
and substrate degraded were taken from the Nitschke and Pastore (2006)
and compared with the proposed model. These data were are also
compared with the prediction of Monods and Tessier model. Compared
to other models, the one proposed in this work gives better fit (Fig. 8).
The biomass values from Zhang et al. (2005) have also been compared
with many other models along with the proposed one and found that the
proposed model provided better match (Fig. 9).This indicate the
robustness of the proposed model in this work as compared to other
available models. .
5.4. Thermodynamic behavior of biodegradation
In order to make the biodegradation successful for industrial appli-
cation, thermodynamic feasibility is the major concern. Fig. 10 shows the
variation of activation energy and pre-exponential factor during the
course of degradation. It has been observed that the activation energy
required for the reaction increases with increase in time. The amount of
energy required to initiate the degradation has almost doubled on 20th
day of degradation when compared to day 1. This is because activity of
bacterial metabolism has found to be reduced over the period of time.
During the course of incubation the microorganism will undergo the
stationary growth phase and dead phase and their activity will reduce.
Hence, the energy required to activate the biodegradation process has
found to be increased with increase in incubation time. The positive
value of Ea indicates that the degradation reaction is endothermic in
nature. Table 5 gives the thermodynamic parameters for the biodegra-
dation of waxy crude oil using Bacillus subtilis. From the results, we have
observed the change in thermodynamic properties of biodegradation
with respect to temperature during the biodegradation process. Gibbs
free energy has also been increased with increase in temperature irre-
spective of the activity of microorganisms. Similarly, the change in
enthalpy of biodegradation has found to decrease with increase in in-
cubation temperature. The negative values of ΔS showed that the for-
mation of the metabolites, such as biosurfactant by Bacillus subtilis is
connected with a decrease in entropy. Also, the negative values of en-
tropy show that the biodegradation activated compound is highly orga-
nized compared to the initial compound. Mulla (2012) reported that
thermodynamic compensation in any reaction occurs because of the
Fig. 10. Variation of activation energy and pre-exponential factor as a function of
degradation time.
N. Sakthipriya et al. Journal of Petroleum Science and Engineering 160 (2018) 412–421

Table 5 Contois, D.E., 1959. Kinetics of bacterial growth: relationship between population density
Thermodynamic parameters for the biodegradation of waxy crude oil using Bacillus subtilis. and specific growth rate of continuous culture. J. Gen. Microbiol. 21, 40–50.
Cooper, D.G., Macdonald, C.R., Duff, S.J.B., Kosaric, N., 1981. Enhanced production of
Degradation period Temperature K ΔH ΔS ΔG surfactin from continuous product removal and metal cation additions. Appl.
Environ. Microbiol. 42, 408–412.
Day 1 308 1390.02 42.89 14 601.00
Darvishi, P., Ayatollahi, S., Mowla, D., Niazi, A., 2011. Biosurfactant production under
323 1263.73 42.50 14 992.06
extreme environmental conditions by an efficient microbial consortium, ERCPPI-2.
348 1055.88 41.88 15 631.08
Colloids Surf. B Biointerfaces 84, 292–300.
Day 5 308 2180.85 30.84 11 680.37 de Oliveira, D.W.F., Franç, I.W.L., Felix, A.K.N., Martins, J.J.L., Giro, M.E.A.,
323 2056.13 30.44 11 890.60 Melo, V.M.M., Goncalves, L.R.B., 2013. Kinetic study of biosurfactant production by
348 1848.28 29.82 12 228.24 Bacillus subtilis LAMI005 grown in clarified cashew apple juice. Colloids Surf. B
Day 10 308 2821.36 26.10 10 861.28 Biointerfaces 101, 34–43.
323 2696.65 25.70 11 000.42 Etoumi, A., 2007. Microbial treatment of waxy crude oils for mitigation of wax
348 2488.80 25.08 11 219.58 precipitation. J. Pet. Sci. Eng. 55, 111–121.
Day 15 308 3744.04 20.33 10 006.83 Ghojavand, H., Vahabzadeh, F., Shahraki, A.K., 2012. Enhanced oil recovery from low
323 3619.33 19.93 10 059.43 permeability dolomite cores using biosurfactant produced by a Bacillus mojavensis
348 3411.48 19.31 10 134.34 PTCC 1696 isolated from Masjed-I Soleyman field. J. Pet. Sci. Eng. 81, 24–30.
Grishchenkov, V.G., Townsend, R.T., McDonald, T.J., Autenrieth, R.L., Bonner, J.S.,
*ΔH-change in enthalpy, J/mol; ΔS-change in entropy, J/mol.K; ΔG-Gibbs free energy Boronin, A.M., 2000. Degradation of petroleum hydrocarbons by facultative
change, J/mol. anaerobic bacteria under aerobic and anaerobic conditions. Process Biochem. 35,
889–896.
Hassanshahian, M., Zeynalipour, M.S., Musa, F.H., 2014. Isolation and characterization of
crude oil degrading bacteria from the Persian Gulf (Khorramshahr provenance). Mar.
linear relationship between entropy and enthalpy, which is in-line with
Pollut. Bull. 82, 39–44.
the results obtained here. Hong, J., 1989. Yield coefficients for cell mass and product formation. Biotechnol. Bioeng.
33, 506–507.
Kumar, A., Kumar, S., Kumar, S., 2005. Biodegradation kinetics of phenol and catechol
6. Conclusion using Pseudomonas putida MTCC 1194. Biochem. Eng. J. 22, 151–159.
Lee, Y.K., 2006. Bioprocess Technology, Microbial Biotechnology: Principles and
This work involves the kinetic modeling of the crude oil biodegra- Applications, second ed. World Scientific publishing Co Pvt. Ltd., Singapore,
pp. 23–72.
dation reaction incorporating microbial growth, biosurfactant produc- Lin, J., Lee, S., Lee, H., Koo, Y., 2000. Modeling of typical microbial cell growth in batch
tion and carbon substrate (crude oil) utilization using Bacillus subtilis YB7 culture. Biotechnol. Bioprocess Eng. 5, 382–385.
a thermophilic organism. It has been observed that the incubation has a Lin, C.S.K., Du, C., Koutinas, A., Wang, R., Webb, C., 2008. Substrate and product
inhibition kinetics in succinic acid production by Actinobacillus succinogenes.
greater impact on the biodegradation and its reaction kinetics. The linear
Biochem. Eng. J. 41, 128–135.
relationship is existed between the surfactin produced and the quantity of Low, S.P., Bada, J.L., Somero, G.N., 1973. Temperature adaptation of enzymes: roles of
waxy crude oil degraded. Various growth models have been explored to the free energy, the enthalpy, and the entropy of activation. Proc. Nat. Acad. Sci. 70,
430–432.
establish the most satisfactory one to define the existing process dy-
Mercier, P., Yerushalmi, L., Rouleau, D., Dochain, D., 1992. Kinetics of lactic acid
namics. Among the various model equations, the Logistic and Contois fermentation on glucose and corn by Lactobacillus amylophilus. J. Chem. Technol.
model gave slightly reasonable fit with the experimental values of Biotechnol. 55, 111–121.
biomass dry weight, biosurfactant production and waxy crude oil Monod, J., 1949. The growth of bacterial cultures. Annu. Rev. Microbiol. 3, 371–394.
Mulla, A.A., 2012. Enthalpy-entropy compensation in polyester degradation reactions.
degradation. However, the model proposed in this work, which is a Int. J. Chem. Engg 2012, 1–8.
modification of the Monod's model, gives the best fit for all the systems Nakhla, G., Liu, V., Bassi, A., 2006. Kinetic modeling of aerobic biodegradation of high oil
studied. The proposed model has also been compared with the other and grease rendering wastewater. Bioresour. Technol. 97, 131–139.
Nitschke, M., Pastore, G.M., 2006. Production and properties of a surfactant obtained
experimental values reported in the literature and is found to give better from Bacillus subtilis grown on cassava wastewater. Bioresour. Technol. 97, 336–341.
fit as compared to any other model. It is believed that this study will be Noordman, W.H., Janssen, D.B., 2002. Rhamnolipid stimulates uptake of hydrophobic
suitable for possible model development for bioremediation in oil and compounds by Pseudomonas aeruginosa. Appl. Environ. Microbiol. 4502–4508.
Ostendorf, D.W., Schoenberg, T.H., Hinlein, E.S., Long, S.C., 2007. Monod kinetics for
gas sectors. aerobic biodegradation of petroleum hydrocarbons in unsaturated soil microcosms.
Environ. Sci. Technol. 41, 2343–2349.
Acknowledgements Pirt, S.J., 1975. Principles of Microbial and Cell Cultivation. Blackwell Scientific
Publications, Oxford.
Prats, M., Rodriguez, F., 1992. A Michaelis-Menten enzyme: time correction introduced
We authors thank the Institute of Oil and Gas Production Technology for computing kinetic parameters at low values of [S]/Km. Biochem. Educ. 20,
(IOGPT), Oil and Natural Gas Corporation (ONGC), Panvel, India for 176–177.
Reed, M.C., Lieb, A., Nijhout, H.F., 2010. The biological significance of substrate
providing the composition and carbon profiling of the original crude oil inhibition: a mechanism with diverse functions. Bioessays 32, 422–429.
sample through the support IOGPT/PROC/SRN: 18/12-13. We also Robinson, J.A., Tiedje, J.M., 1983. Nonlinear estimation of Monod growth kinetic
thank SAIF (IIT Madras) for helping in analyzing the composition of the parameters from a single substrate depletion curve. Appl. Environ. Microbiol. 45,
1453–1458.
waxy crude oil and model oil.
Sakthipriya, N., Doble, M., Sangwai, J.S., 2015a. Biosurfactant from Pseudomonas species
with waxes as carbon source - their production, modelling and properties. J. Ind. Eng.
References Chem. 31, 100–111.
Sakthipriya, N., Doble, M., Sangwai, J.S., 2015b. Fast degradation and viscosity reduction
of waxy crude oil and model waxy crude oil using Bacillus subtilis. J. Petrol. Sci. Eng.
Ahmadi, E., Yousefzadeh, S., Taghipour, N., 2017. Performance, kinetic, and
134, 156–166.
biodegradation pathway evaluation of anaerobic fixed film fixed bed reactor in
Sakthipriya, N., Doble, M., Sangwai, J.S., 2015c. Action of biosurfactant producing
removing phthalic acid esters from wastewater. Sci. Rep. 7, 1–14.
thermophilic Bacillus subtilis on waxy crude oil and long chain paraffins. Int. Biodeter.
Al-Bahry, S.N., Al-Wahaibi, Y.M., Elshafie, A.E., Al-Bemani, A.S., Joshi, S.J., Al-
Biodegr 105, 168–177.
Makhmari, H.S., Al-Sulaimani, H.S., 2013. Biosurfactant production by Bacillus
Salehizadeh, H., Mohammadizad, S., 2009. Microbial enhanced oil recovery using
subtilis B20 using date molasses and its possible application in enhanced oil recovery.
biosurfactant produced by Alcaligenes faecalis. Iran. J. Biotechnol. 9, 216–223.
Inter. biodeter. Biodegr 81, 141–146.
Schmidt, S.K., Simkins, S., Alexander, M., 1985. Models for the kinetics of biodegradation
Al-Wahaibi, Y., Joshi, S., Al-Bahry, S., Elshafie, A., Al-Bemani, A., Shibulal, B., 2014.
of organic compounds not supporting growth. Appl. Environ. Microbiol. 50, 323–331.
Biosurfactant production by Bacillus subtilis B30 and its application in enhancing oil
Simkins, S., Alexander, M., 1984. Models for mineralization kinetics with the variables of
recovery. Colloids Surf. B Biointerfaces 114, 324–333.
substrate concentration and population density. Appl. Environ. Microbiol. 47,
Bao, M., Wang, L., Sun, P., Cao, L., Zou, J., Li, Y., 2014. Biodegradation of crude oil using
1299–1306.
an efficient microbial consortium in a simulated marine environment. Mar. Pollut.
Sivakumar, A., Srinivasaraghavan, T., Swaminathan, T., Baradarajan, A., 1994. Extended
Bull. 64, 1177–1185.
Monod kinetics for substrate inhibited systems. Bioprocess Eng. 11, 185–188.
Choi, J.W., Rim, Y.K., Lee, W.H., Henrik Pedersen, H., Chin, C.K., 1999. Kinetic model of
Tehrani, D.M., Herfatmanesh, A., 2015. Biodegradation of aliphatic and aromatic
cell growth and secondary metabolite synthesis in plant cell culture of Thalictrum
fractions of heavy crude oil–contaminated soil: a pilot study. Biorem. J. 11, 71–76.
rugosum. Biotechnol. Bioprocess Eng. 4, 129–137.

420
N. Sakthipriya et al. Journal of Petroleum Science and Engineering 160 (2018) 412–421

Tessier, G., 1936. Quantitative laws of growth. Ann. Physiol. Physicochim. Biol. 12,
527–573.
Vaz, D.A., Gudina, E.J., Alameda, E.J., Teixeira, J.A., Rodrigues, L.R., 2012. Performance
of a biosurfactant produced by a Bacillus subtilis strain isolated from crude oil samples
as compared to commercial chemical surfactants. Colloid. Surf. B 89, 164–174.
Zhang, L., Wu, Y.T., Qian, X.P., Meng, Q., 2005. Biodegradation of crude oil by
Pseudomonas aeruginosa in the presence of rhamnolipids. J. Zhejiang Univ. Sci. B 6,
725–730.

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