The hydrocarbon industry has reached a considerable growth during
the last decades, and nowadays, it is one of the most essential links in
the economic and social development worldwide. The increase in total
demand for energy and water has led to the implementation of water-

intensive forms of power generation and energy-intensive platforms
of water production, primarily driven by population growth.
Consequently, this excessive energy consumption has triggered the
frequent exploitation of hydrocarbon reserves without considering
the environmental impacts on terrestrial, aquatic and aerial
ecosystems (Leahy and Colwell, 1990). The limited biodegradation
capacity of petroleum-derived hydrocarbons and their low reactivity
represent a significant threat to the environment, owing to the high
level of toxicity and inhibition to plant and animal growth and their
mutagenic and carcinogenic characteristics (United Nations
Educational Scientific and Cultural Organization, 2019). The above
statement agrees with what Adams et al. (2008) affirm: "Oil
contamination in bodies of water causes an impermeable film that
quickly affects gas exchange and the passage of sunlight, giving way to
the rupture of the food chain and a series of simultaneous physical
and chemical changes, which make the natural degradation process
slower, inefficient, and toxic”. This can produce substantial structural
changes in the phytoplankton communities and the rest of marine
fauna and flora (Asimea and Sam-Wobo, 2011).
On average, roughly nine million tons of petroleum hydrocarbons are
discharged into aquatic ecosystems all over the world each year,
especially into marine waters and estuaries (Torres, 2003). Indeed,
the oil spillage in the Gulf of Mexico, considered one of the most
catastrophic events in history, released about 600000 tons of crude oil
into the sea (Dell’Anno et al., 2018). The largest source of pollution by
hydrocarbons and their derivatives in marine environments comes
from routine ship/boat washing activities, natural oil leaks on the sea
surface, and accidents during the exploration and transportation of
crude oil (Marques-Junior et al., 2009).
Although conventional oil removal methods such as physical
extraction are often the first response option, they unlikely achieve a
complete cleanup of oil spills. These techniques often use traditional
physical methods such as grease traps, evaporation, and separation
with ultrafiltration membrane. Additionally, chemical methods like
gas and ozone injection, chemical precipitation, ion exchange are
usually applied to treat this type of contamination. The problem is that
these methods require high investment for their implementation and

operation, and in some cases, end up transferring pollutants to other
media.
The negative impacts on the environment, food safety, human health,
the integrity of fauna and flora species, and the stability of petroleum
hydrocarbons make it necessary to develop alternative treatment
methods to the physical and chemical methodologies. These must be
more effective, environmentally friendly, and faster compared to
natural biodegradation processes.
Bioremediation processes are an alternative technology that meets
these requirements and have been on the rise since the early 1990s
when they were popularized as the ultimate solution to oil spills (Hoff,
2003). This technology seeks to recover contaminated sites using
organisms (plants, fungi, bacteria, or enzymes). For this purpose, it
considers the metabolic processes of the microorganisms and how
they will transform the pollutant into biomass and carbon dioxide
(mineralization) (Gamba and Pedraza, 2018). This biological
remediation uses bio-stimulation and bio-augmentation as potential
strategies to hasten natural attenuation or biodegradation (Ladousse
and Tramier, 1991). The last advances in sustainable technologies
have led to the use of surfactants, which are chemical compounds with
high surface activity (Gervajio et al., 2020). They can improve the
conditions and results of bioremediation. Bio-surfactants are a kind of
surfactant naturally produced by microorganisms or extracted from
plants or animals. Owing to their biodegradability and low toxicity,
they are preferred to remediate petroleum hydrocarbon-
contaminated sites (Silva et al., 2014).
The possibility of synthesizing this type of compound from low-cost
sources and industrial waste has made them ideal for treating areas
affected by oil. Furthermore, their outstanding biodegradation
capability, detoxification of industrial effluents, and high effectiveness
under conditions of extreme temperature, pH, and salinity manifest
their versatility (Pirôllo, 2006). Moreover, the addition of surfactants
is paramount during the preparation of hybrid nanofluids since they
can improve the thermophysical and rheological properties of this
type of nanofluids. The application of them enables the longer stability
period of hybrid nanofluids with an uniform dispersion of

nanoparticles, increasing the thermal conductivity and decreasing the
viscosity (Shah and Koten, 2020). These characteristics have driven to
a significant production of natural surfactants, as supported by data
from Campos et al. (2013), who point out that in 2012 these
compounds represent 3.5 million tons of the total of surfactants
produced worldwide, which is translated into the generation of 6588
million dollars per year.
The importance of this project, developed on a laboratory scale, lies in
analyzing the bio-catalytic agent quality through the monitoring of
degradation behavior of hydrocarbons present in seawater as a
function of the Total Petroleum Hydrocarbon Content (TPHC) and
some physical and chemical parameters such as Chemical Oxygen
Demand, Dissolved Oxygen, turbidity, among others.

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2. Materials and methods
2.1. Chemicals
All reactants used were technical, analytical, pure, or reagent grades
without being modified in their original composition. Hydrochloric
acid (HCl, 37 wt.%), glycerol (C 3 H 8 O 3,  99 wt.%), sodium dodecyl sulfate
(SDS) (NaC 12 H 25 SO 4,  90 wt.%), ethyl alcohol (C 2 H 5 OH, 99 wt.%), and
methanol (CH 3 OH, 99 wt.%) were provided by Labsynth. β-
mercaptoethanol (C 2 H 6 OS, 99 wt. %), tris (hydroxymethyl)
aminomethane-HCl (CNH 2 (CH 2 OH) 3,  99 wt.%), bromophenol blue
(C 19 H 10 Br 4 O 5 S, 99 wt.%), acrylamide (C 3 H 5 ON, 99 wt.%), tris(2-
carboxyethyl) phosphine (C 9 H 15 O 6 P, 99 wt.%), formic acid (CH 2 O 2,  95
wt.%), sodium form (NaCOOH, 99 wt.%), potassium acid phthalate
(C 8 H 5 KO 4 ), and silver nitrate (AgNO 3,  98 wt.%) were sold by Sigma-
Aldrich. Ethylenediaminetetraacetic acid (EDTA) (C 10 H 16 N 2 O 8,  99 wt. %)
and kerosene (C 12 H 26,  99 wt. %) were provided by Neon and Natrielli,
respectively. Potassium dichromate (K 2 Cr 2 O 7,  99.5 wt. %), mercury
sulfate (Hg 2 SO 4,  98 wt. %), silver sulfate (Ag 2 SO 4,  99 wt.%), and
potassium chromate (K 2 CrO4, 99.5 wt.%) were purchased from
PanReac. Sulfuric acid (H 2 SO 4,  95–98 wt. %) and hexane (C 6 H 14,  ≥ 98.5
wt. %) were obtained from J.T.Baker.

2.2. Sample preparation and bioremediation study
The oil-polluted water used in this study was obtained by spilling a
specific volume of Vasconia heavy crude oil on natural seawater. The
crude oil sample was collected from an oil & gas refining company in
Colombia, and the seawater was obtained directly from the sea in a
coastal town in Colombia. The properties of the crude oil sample were:
API gravity (24.27 API°), specific gravity at 15 °C (0.908), viscosity at
40 °C (22 cSt), Sulphur content (0.833 wt.%), Reid Vapor Pressure
(RVP) (21.99 kPa), and Flash Point (0 °C).
The bio-catalyst was supplied by its official distributor in Colombia.
The product composition comprises sodium benzoate, imidazolidinyl
urea, diazolidinyl urea, a fermentation supernatant derived from a
Saccharomyces cerevisiae culture, and a non-ionic surfactant that was
extracted from plants and minerals. It can belong to, but is not limited
to, polyether non-ionic surfactants comprising fatty alcohols, alkyl
phenols, fatty acids, and fatty amines which have been ethoxylated;
polyhydroxyl non-ionic (polyols) typically comprising sucrose esters,
sorbital esters, alkyl glucosides, and polyglycerol esters which may or
may not be ethoxylated (Dale and Hill, 1998). According to the
supplier, this product is a totally safe and completely soluble in water
bio-catalytic degrader of organic waste materials. This bio-catalyst is
biodegradable regarding the positive results of the Organization for
the Economic Co-operation and Development (OCDE) 302B test for
ready biodegradability. Also, eco-toxicity characteristics were tested
for microorganisms and aquatic organisms on an acute basis
((LC 50 /EC 50  between 1 and 10 mg/L in the most sensitive species
tested). These results validate the non-toxicity nature of this
product(Neozyme International, 2015).
The crude oil-polluted seawater samples were prepared into six
beakers by adding 0.5 mL of Vasconia crude oil to 700 mL of seawater
in each glass vessel. The seawater bottles were stored in six translucid
glass vessels and then left to stand for four (4) days to allow the
indigenous micro-organisms to acclimatize to their new
environment. Table 1 shows the various samples and their
constituents.

Table 1
Samples used and their components.
Sample Components
A
(Control) Crude oil and seawater only (640 mg L −1 )
B Seawater, bio-catalytic agent solution (2167.39 mg L −1 ), and crude oil (640 mg

L −1 )

C Seawater, bio-catalytic agent solution (10000 mg L −1 ) and crude oil (640 mg

L −1 ).

D
(Control) Seawater, bio-catalytic agent solution (2167.39 mg L −1 ) and aeration system.
E Seawater, bio-catalytic agent solution (2167.39 mg L −1 ), crude oil (640 mg L −1 )

and aeration system.

F Seawater, bio-catalytic agent solution (10000 mg L −1 ), crude oil (640 mg L −1 )

and aeration system.

Open in a separate window
Samples labeled B, D, and E were amended by the addition of 1.3 mL
of bio-catalyst solution concentrated at 2167.39 mg L −1  (bio-catalyst)
to each mixture of the samples, following the instructions stated on
the product data-sheet. On the other hand, the same volume of bio-
catalytic agent solution but with a concentration of 10000 mg L −1  was
added to samples labeled C and F to evaluate the effect of the dosage
of the bio-catalytic agent on the effectiveness of the bioremediation
process. This concentration was selected regarding the information
provided by the manufacturing company of the product. They state
that this bio-catalyst can be applied to TPH contaminated soil,
shorelines, and beaches at dilutions of 0.2%–2% v/v.
All the experimental set-up vessels were stored at 25 °C and average
relative humidity of 64.5%. During the incubation time, the
temperature and relative humidity percentages were continuously
controlled by a sensor (PCE Instruments, PCE-P18L, and model). The
samples labeled D, E and F were agitated uninterruptedly for aeration
and mixing to increase contact between the indigenous microbial
consortium, nutrients, and contaminated water.
The other samples labeled A, B, and C were subjected to an agitation
system in a magnetic stirrer. Samples from each vessel were analyzed

on days 0,4,9,16,23, and 30. The following bioremediation indicating
parameters in the polluted water were monitored in the study of
remediation; Chemical Oxygen Demand (COD), Turbidity, Total
Petroleum Hydrocarbon Content (TPHC), pH, and Dissolved Oxygen
(DO).
Considering that the growth of aerobic mesophylls microorganisms is
propitious in polluted and aerated medium, the Total Microbial Count
(TMC) was measured only on the crude oil-contaminated samples
with an aeration system (E and F). Likewise, it is worth pointing out
that due to the absence of external bacteria consortium (bio-
augmentation), the measurements of TMC were only taken at the
beginning and the end of the experiment to observe a significant
difference in the bacterial growth.
2.3. Methods used in analytical studies
The following methods were quite relevant to determine the bio-
catalyst quality and predict its more possible degradation behavior.
The qualitative characterization was the criteria to decide to analyze
the physicochemical properties of the seawater and confirm the
expected performance of the product.
2.3.1. Drop-collapse test
This test was executed according to the experimental method
described by Jain et al. (1991), and adapted by Bodour and Miller-
Maier (1998). A clean flat surface was used to carry out the
experiment, and the holes in there were filled with 5 μL of vegetable
oil and 5 μL of bio-catalyst solution were added to the oil surface.
After that, the behavior of the drop was inspected for 1 min. If the
drop retains its shape, it indicates a negative result, while if the drop
collapses mean a positive response.
2.3.2. Oil-spreading assay
5 mL of distilled water were poured into a 15 cm diameter Petri dish,
followed by the addition of 100 μL of Bazu oil, supplied by a Brazilian
refinery company, to the surface of the water to form a thin layer of
oil. About 10 μL of the bio-catalytic agent solution was carefully added

to the center of the formed oil layer, and the diameter of the cleaned
area was measured (Moro et al., 2018). If the action of bio-catalytic
agent is significant, the oil layer will be displaced, resulting in a
decontaminated zone free of crude oil. The diameter measurement is
closely related to the surfactant activity (Pornsunthorntawee et al.,
2008).
2.3.3. Emulsification assay (E 24 )
The emulsifying activity of the studied product was measured using
the method described by Cooper and Goldenberg (1987). The test was
realized by mixing 2 mL of kerosene with an equal volume of a bio-
catalytic agent, which was previously stirred in a vortex type agitator
for 2 min and left to stand for twenty-four (24) hours. The
emulsification index was calculated as the ratio between the height of
the emulsion of foam layer (cm) and the total height of the liquid in
the tube (cm), multiplied by 100.
2.3.4. Critical Micelle Concentration (CMC)
This test required the preparation of different dilutions of a bio-
catalytic agent in distilled water. The changes in surface tension were
measured in a Gibertini brand digital tensiometer at a temperature of
25 °C (Moro et al., 2018). The CMC value of the bio-catalytic agent was
determined graphically by the surface tension inflection point (Y-axis)
versus the bio-catalytic agent concentration (X-axis) (Nitschke and
Pastore, 2006).
The tensiometer measurements were taken by immersion of a
coverslip below the surface of the surfactant solution (1 mm
approximately), which was slowly extracted, then the maximum force
was measured and registered. The distilled water of 96% purity was
used as standard.
2.3.5. Determination of pH
The pH values were obtained using a pH and temperature probe
(HACH® HQ40D, model) coupled with a multiparameter of
continuous reading that worked under the potentiometric method
described by the 4500-H APHA Standard Method (American Health

Public Association, 2017a). The uncertainty associated with the
equipment reading was ±0.016(Standard Deviation).
2.3.6. Determination of turbidity
The Standard Methods 2130 B protocol (American Health Public
Association, 1992) was followed to analyze the turbidity during the
remediation time. The turbidity of the samples was determined using
a turbidimeter (TN400/TUR-001) suitable for readings between 0.02 –
800 NTU. The uncertainty associated with the equipment reading was
±0.053(Standard Deviation).
2.3.7. Determination of dissolved oxygen
The Dissolved Oxygen was measured using a luminescence Oxygen
probe (HACH® (LDO) LDBO101, model), coupled with a
multiparameter with continuous reading. The dissolved Oxygen
content values were in the range of 0.01 up to 20 mgO 2  L −1 . The
uncertainty associated with the equipment reading was
±0.048(Standard Deviation).
2.3.8. Determination of Chemical Oxygen Demand
COD concentration was determined by the spectrophotometric
method 5220D (closed reflux spectrophotometric method), described
in the Standard Methods for Examination of Water and Wastewater
(American Health Public Association, 2017b).
According to the procedure, the digestion tubes were prepared by
adding 1.5 mL of digestion solution and 3.5 mL of catalyst solution.
They were left in agitation for two (2) days until complete dissolution.
Thenceforth, 2.5 mL of sample was added to the test tube and
hermetically sealed, with subsequent agitation for the homogenization
of all components inside the digestion tube. The tubes were taken to a
thermoreactor (HANNAH Instruments® 839800, model) for two (2)
hours at 150 °C. After the digestion process, the tubes were removed
from the thermoreactor and get cold up to room temperature.
Hereafter, the COD concentrations were taken in Oxygen mg L −1  using a
spectrophotometer (GENESYS™ 10S UV VIS, model). The uncertainty
associated with the equipment reading was ±0.125(Standard
Deviation).

2.3.9. Determination of Total Petroleum Hydrocarbon Content (TPHC)
The TPHC values were measured and monitored according to the
gravimetric method 1664A of the USEPA (The United States
Environmental Protection Agency), which involves a liquid-liquid
extraction with hexane followed by the concentration of Total
Petroleum Hydrocarbon (TPH) in a roto-evaporator system. The
results of this analysis were obtained as the difference between the
final weight of the flask and the initial weight of the dry and empty
flask in mg, divided by the initial volume of the sample in Litres
(United States Environmental Protection Agency, 2000). The
uncertainty associated with the equipment reading was
±0.015(Standard Deviation).
The Chemical Oxygen Demand and the TPHC were defined as the
following parameters: COD percentage removal efficiency (%CODR)
and Total Petroleum Hydrocarbon Percentage Removal (%TPHR),
respectively (See Eqs. (1) and (2)).

%RCOD=(CODi−CODfCODi)∗100

(1)

where %RCOD is the percentage of Chemical Oxygen Demand
Removal Efficiency; COD f,  is the COD concentration (ppm) at the end of
the experiment, and COD i,  is the COD concentration (ppm) at the
beginning of the experiment

%RTPH=(TPHi−TPHfTPHi)∗100

(2)

where %RTPH is the percentage of TPH clean-up or removal
efficiency; TPH f  is the TPH concentration (ppm) at the end of the
experiment, and TPH i  is the TPH concentration (ppm) at the beginning
of the experiment.
2.3.10. Determination of TMC

The count of aerobic mesophilic microorganisms was performed
according to the NTC 4519 method (Instituto Colombiano de Normas
Técnicas y Certificación (ICONTEC), 2013). To quantify the viable
microorganisms, the sample was inoculated in a culture medium and
poured into a Petri dish. An automatic spiral plater (NF V08-100) was
used to incubate the sample at 35 °C for seventy-two (72) hours. The
TMC values were collected from the number of colonies counted in the
Petri dish per 100 mL of sample.

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3. Results and discussions
3.1. Qualitative characterization of the bio-catalytic agent
Drop-collapse test and oil-spreading assay are qualitative and fast
tests used for the prior evaluation of the surface activity of the bio-
catalytic agent and its performance. Both of them tested a positive
result indicating a satisfactory efficiency of the product. The evident
cleaning and removal action of petroleum from the contaminated area
(Petri dish) is intimately associated with the amphiphilic nature of the
non-ionic surfactant present in this bio-catalyst, which reduces the
surface tension and favored the miscibility between two different
polarities substances such as water and oil.
3.2. Emulsification assay (E 24 )
Franzetti et al. (2010) reported that the emulsification process begins
when there is enough accumulation of surfactant that forms a solution
that contains tiny droplets of oils suspended in an aqueous
medium. Figure 1 shows the set-up of the experiment carried out to
evaluate the emulsifying activity of the bio-catalyst. After twenty-four
(24) hours, the average emulsification index obtained from three
independent measurements was 74.47% ± 5.55 as proof of the
excellent tensoactive properties of the bio-catalyst studied. This value
surpasses the acceptable emulsification index for a good surfactant of
40%, which buttresses the high product quality (Youseff et al., 2004);
additionally, the high molecular weight of surfactants gives them the
characteristic of efficient emulsifiers (Souza et al., 2014).

Figure 1
Test-tube used to measure the emulsification index (E 24 ), (1). The first test-tube is the
control sample whose composition is only 2 mL kerosene. (2), (3), (4). These test-tubes
contain a mixture of 2 mL kerosene with 2 mL of bio-catalytic agent solution previously
stirred in a vortex type agitator for 2 min.
3.3. Critical Micelle Concentration
By means of this test was possible to determine the CMC of the bio-
catalytic agent evaluated in this study. This concentration is known as
the value from which begins the micelle formation (Souza et al., 2014).
The minimal concentration of surfactant required to reduce the
surface tension to its maximum extension, enhancing the oil solubility
in the aqueous medium (Moro et al., 2018). As presented in Table 2,
approximately 40 mg L −1  of bio-catalytic agent solution was necessary
to reduce the surface tension of water from 73.1 to 29.0 mN m −1 . These
outcomes are comparable to the CMC values obtained for the most

efficient surfactant tested by Moro et al. (2018). They also observed a
high initial value of surface tension corresponds to water, followed by
a significant decrease due to the presence of bio-catalyst in the
solution. The CMC was then selected at the minimum value of surface
tension obtained, henceforth the surface tension measurements were
kept almost constant regardless of the increase in the concentration of
bio-catalytic solution. These results render attractive this bio-catalytic
agent, knowing that a surfactant is considered suitable when it is able
to reduce the surface tension of water to 35 nN m −1  or less (Patowary
et al., 2015). Zhang and Miller (1992) mention that the required
concentration to diminish the surface tension of water from 71.2 mN
m −1  to values below 40 mN m −1  varies between 1 and 200 mg L −1 . The
CMC of this surfactant is low, which means that a low concentration of
the product can decrease the surface tension of water, favoring the
biological availability of the hydrophobic substrate (petroleum
hydrocarbons) to microorganisms and the interfacial surface
reduction between the bacteria cell wall and hydrocarbon molecules.
Table 2
Surface Tension values measured at a given concentration of bio-catalytic agent.
Bio-catalytic Agent Concentration (mg L −1 ) Surface Tension (mN m −1 )
540 30.0 ± 0.08
430 30.2 ± 0.09
140 29.8 ± 0.16
70 29.4 ± 0.25
60 29.6 ± 0.11
50 29.8 ± 0.41
40 29.0 ± 0.10
30 29.2 ± 0.64
20 29.8 ± 0.70
10 29.2 ± 1.05
5 29.4 ± 1.23
0 (Distilled water without bio-catalyst nor oil) 73.1 ± 0.95

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The concentrations tested in this study were above the CMC. This is
based on some findings that conclude that the surfactant

concentration must be above the respective CMC, to increase the
solubilization/desorption of aliphatic or Polycyclic Aromatic
Hydrocarbons (PAHs) from one medium to another (Zhu and Aitken,
2010), and achieve the maximum effect of the surfactant (Sajjadi et al.,
2010).
3.4. Analysis of chemical and physical properties of seawater
during the bioremediation
Figure 2 illustrates the variation of Dissolved Oxygen (DO) with
respect to the remediation time for the polluted water sample in the
six vessels. It was appreciated that the DO increased with remediation
time for the samples labeled D, E, and F, where took place an effective
degradation associated with the cracking of petroleum hydrocarbons.
Hence, the indigenous microorganisms present in the medium do not
need the same Oxygen concentration for their respiration process due
to the reduction of organic matter.

Figure 2
Variation of dissolved oxygen content with remediation time. (A) Seawater with oil (640
mg L −1 ); (B) Seawater with oil (640 mg L −1 ) and bio-catalytic agent solution (2167 mg L −1 );
(C) Seawater with oil (640 mg L −1 ) and bio-catalytic agent solution (10000 mg L −1 );

(D)Seawater with bio-catalytic agent solution (2167.39 mg L −1 ) and aeration system; (E)
Seawater with oil (640 mg L −1 ), bio-catalytic agent solution (2167 mg L −1 ), and aeration
system; (F) Seawater with oil (640 mg L −1 ), bio-catalytic agent solution (10000 mg L −1 )
and aeration system.
The least DO value was observed in the control sample(A), which did
not have the bio-catalyst solution or the aeration system. Likewise, the
samples labeled B and C showed the same behavior as the sample
labeled A. However, it is worth pointing out that after day sixteen (16)
of the experiment, the DO content started rising in the samples labeled
B and C. This event is related to bio-catalytic agent capacity to
improve the Oxygen transfer and speed up the degradation of organic
matter. The higher the Dissolved Oxygen level, the better the water
quality and vice versa.
Turbidity analysis displayed positive results for oil-polluted samples
labeled E and F stimulated with the abio-catalytic agent solution and
an aeration system. They achieved turbidity reduction values of
61.357% ± 0.053 and 79.623% ± 0.053, respectively (see Figure 3).
Turbidity is inversely proportional to water quality. Therefore, it is
valid to affirm that the sample labeled F displays better quality and
performance than other studied samples during the remediation time.

Figure 3
Variation of turbidity with remediation time. (A) Seawater with oil (640 mg L −1 ); (B)
Seawater with oil (640 mg L −1 ) and bio-catalytic agent solution (2167 mg L −1 ); (C)
Seawater with oil (640 mg L −1 ) and bio-catalytic agent solution (10000 mg L −1 ); (D)
Seawater with bio-catalytic agent solution (2167.39 mg L-1) and aeration system; (E)
Seawater with oil (640 mg L −1 ), bio-catalytic agent solution (2167 mg L −1 ), and aeration
system; (F) Seawater with oil (640 mg L −1 ), bio-catalytic agent solution (10000 mg L −1 )
and aeration system.
From Figure 4, it was observed that samples labeled A, B, C, D, and E
showed a similar tendency. In the beginning, was detected a decrease
in the pH values linked to the possible decomposition of petroleum
hydrocarbons to carbon dioxide. Nonetheless, from sixteen (16) days,
these samples started increasing its pH value as proof of
bioremediation (Anih et al., 2019). The pH of the sample labeled F
exhibited a continuous rising in pH values, indicating that pollutant
(petroleum hydrocarbons) in the water was decomposed to
compounds that are more basic and less toxic (Amenaghawon et al.,
2014; Obahiagbon and Aluyor, 2009).

Figure 4
Variation of pH with remediation time. (A) Seawater with oil (640 mg L −1 ); (B) Seawater
with oil (640 mg L −1 ) and bio-catalytic agent solution (2167 mg L −1 ); (C) Seawater with oil
(640 mg L −1 ) and bio-catalytic agent solution (10000 mg L −1 ); (D) Seawater with bio-
catalytic agent solution (2167.39 mg L-1) and aeration system; (E) Seawater with oil
(640 mg L −1 ), bio-catalytic agent solution (2167 mg L −1 ), and aeration system; (F)
Seawater with oil (640 mg L −1 ), bio-catalytic agent solution (10000 mg L −1 ) and aeration
system.
The effect of remediation time on TPHC of the samples is presented
in Figure 5. From this graphic is possible to evidence the TPHC
decrease with remediation time for samples labeled E and F. This
reduction is credited to the presence of a bio-catalyst solution and an
aeration system which generates the formation of micro-bubble.
These micro-bubbles are the result of aggregates of surfactant
molecules with a loose molecular packing, which provokes a more
favorable Oxygen mass transfer into an aqueous medium (Basařová
and Zedníková, 2019). This phenomenon contributes to hasten the
crude oil hydrocarbons biological degradation rates.

Figure 5
Variation of total petroleum hydrocarbon concentration with remediation time. (A)
Seawater with oil (640 mg L −1 ); (B) Seawater with oil (640 mg L −1 ) and bio-catalytic agent
solution (2167 mg L −1 ); (C) Seawater with oil (640 mg L −1 ) and bio-catalytic agent
solution (10000 mg L −1 ); (E) Seawater with oil (640 mg L −1 ), bio-catalytic agent solution
(2167 mg L −1 ), and aeration system; (F) Seawater with oil (640 mg L −1 ), bio-catalytic
agent solution (10000 mg L −1 ) and aeration system.
The effect of incorporation micro-bubbles was positive for the
remediation of diesel-contaminated soil carried out by Ayele et al.
(2020). They evaluated the influence of aeration system on diesel-
pollutant removal efficiency and compared it with a non-aeration
system. They noticed that the aeration doubtless help to accelerate the
contaminants degradation rates. With aeration, they achieved to
increase the diesel removal efficiency from 12% to 25% depending on
the particle size of soil, organic matter level, and age of contamination.
This wide gap of removal efficiency could be explained by taking into
account that as airflow rate increases more hydrophobic micro-
bubbles will be created with the large interfacial surface area, which
will enhance the contact surface area between the pollutants and the
surfactant solution helping it to separate pollutants from the
contaminated site (Agarwal and Liu, 2017).

Furthermore, Parhizcar et al. (2015) in their investigation comment
that non-ionic surfactants produce more stable and smaller bubbles
than anionic or cationic surfactants. This is linked to the presence of a
larger hydrophilic group and therefore, the wettability of the channel
wall surface is affected differently. This triggers the more efficient
absorption onto hydrophobic surfaces than onto hydrophilic ones
(Rosen and Kunjappu, 2012).
The analysis of the variation of TPHC for the control sample (A), which
had neither the bio-catalyst solution nor aeration system, allowed us
to observe low TPH concentration values at the beginning of the
experiment due to the absence of the bio-catalyst solution in the
sample. This led to no reduction of surface tension between
petroleum-seawater, affecting the miscibility between the phases and
the emulsion formation. Afterward, the measured concentrations of
TPH trend towards increasing.
On the other hand, samples labeled B and C, in the beginning,
exhibited an increasing tendency of TPHC. However, until sixteen-day,
a TPHC reduction pattern for sample B was identified, while for
sample C was clear to detect an initial decreasing value followed by a
slight increase of TPH concentration. If the experiment had continued,
it would be expected to see a diminishing of TPHC in the sample
labeled B and values below 72.2 mg L −1  (last registered value) for
sample labeled C.
The increase in the concentration of petroleum hydrocarbons for
samples labeled B, C and A is associated with the acclimatization or
adaptation time required by the autochthonous microorganisms due
to the nutrients (bio-catalyst) and contaminants (crude oil) added. It
is presumably that these samples have required much more time than
samples labeled E and F to assimilate the new environment, especially
control sample (A) that did not have bio-catalyst. This, in turn,
obstacle the degradation process. Also, another but less probable
reason could have been the evaporation of water due to a failure in the
hermetic sealing of the vessels.
It should be pointed out that bioremediation of petroleum
hydrocarbons-polluted ecosystems is usually limited due to the

narrow diversity of autochthonous microflora and the scarcity of
specific indigenous microbes for each type of petroleum hydrocarbons
(Ron and Rosenberg, 2014). This event is sharply related to the
behavior of the samples above mentioned, regarding the influence of
the petroleum hydrocarbons-degrading microorganisms on crude oil
removal efficiency. According to Atlas, 1991a, Atlas, 1991b, the
quantity of petroleum-degrading microorganisms in a non-polluted
medium comprises less than 0.1% of the total population. Despite
that, this percentage could ascend to 10% of the total population in
petroleum-contaminated ecosystems, even if it represents the
decreasing microbial diversity of the natural environment (Atlas,
1991b; Gorvanyov, 2015).
This information could be validated when the aerobic mesophilic
bacteria were counted in the aerated samples labeled E and F. Before
contaminating the samples, the quantification method displayed
values below the quantification limit, but at thirty (30) day of the
experiment, the result obtained was 12 CFU 100 mL −1 .
As we expected, the stimulated sample labeled F reached the highest
%RTPH with a rough value of 81.537 ± 0.015, followed by 74.446 ±
0.015 corresponding to sample labeled E. These values are in the
average range of efficiencies reported by Anih et al. (2019), whose
investigation aimed to study the effect of nutrients on bioremediation
of polluted crude-oil water. The results showed that the least TPHC
removal efficiency was 66.1% for the control sample which had
neither nutrients nor microbes added to it, and the highest TPHC
reduction was achieved by a sample that comprised NPK fertilizer as
the bio-catalyst, external microbes and was subjected to an aeration
system. Furthermore, it is quite important to mention that Dell’Anno
et al. (2018), based on the study done by Cheng et al. (2004), suggest
that the combined use of chemical surfactants and bio-catalytic agents
produce a symbiotic effect which can improve the toxic hydrocarbons
removal efficiencies, including those more complicated to degrade as
PAHs. In our case study, this product contains a non-ionic surfactant
from plants, which could explain the rapid and effective TPH removal
from the studied matrices of seawater.

For samples labeled B and C, there were no positive values of
petroleum hydrocarbon removal for samples labeled B and C,
considering their composition and experimental conditions, except for
days twenty-three (23) and thirty (30), when sample labeled B
accomplished an average percentage removal of 19.706% ± 0.015.
The %RTPH could have been improved by applying the bio-
augmentation method and even better bio-stimulation and bio-
augmentation approaches simultaneously. The addition of nutrient
and scarce co-substates to stimulate the existing microorganisms and
bringing new individual strain of microorganism or consortium of
microbial strains in the medium can increasingly boost the
bioremediation results. Many researchers have proven that an array
of a strain of microorganisms is more potential than individual
cultures for metabolizing/degrading a complete group of
hydrocarbons (Deppe et al., 2005; Deziel et al., 1996; Varjani et al,
2013, 2015).
In the case of petroleum which is a mixture of complex and
straightforward hydrocarbons, its simpler compounds can be
degraded by a wide variety of bacteria, but the ability to degrade
complex compounds (such as PAHs, resins, and asphaltenes) is found
in very few species (Varjani, 2017). That is why a bacterial sp.
specializes in the utilization of few hydrocarbons as a preferred food
source while the consortium gives a synergistic effect (Perussitti et al.,
2003; Sugiura et al., 1996; Varjani et al., 2015, 2013). This technique
has achieved TPH removal efficiency values where the difference
grows from 95.54% to 99.09% (Anih et al., 2019).
The COD removal efficiency of studied samples is depicted in Figure 6.
All the seawater matrices showed a similar trend to the variation of
TPH concentration with remediation time. The addition of bio-catalyst
as a source of nutrients and the indigenous microbe consortium
proved to be efficient enough to biodegrade aliphatic petroleum
hydrocarbons, which are the most abundant compounds in the crude
oil used in this study.

Figure 6
Percentage of Chemical Oxygen Demand removal (A) Seawater with oil (640 mg L −1 ); (B)
Seawater with oil (640 mg L −1 ) and bio-catalytic agent solution (2167 mg L −1 ); (C)
Seawater with oil (640 mg L −1 ) and bio-catalytic agent solution (10000 mg L −1 );
(D)Seawater with bio-catalytic agent solution (2167.39 mg L-1) and aeration system;
(E) Seawater with oil (640 mg L −1 ), bio-catalytic agent solution (2167 mg L −1 ), and
aeration system; (F) Seawater with oil (640 mg L −1 ), bio-catalytic agent solution (10000
mg L −1 ) and aeration system.
The highest %RCOD was 64.539 ± 0.125, and it was attained by
sample labeled F, which had the most concentrated bio-catalyst
solution, followed by sample labeled E which had a removal efficiency
of 35.325% ± 0.125 for COD at the end of the remediation time. These
results suggest the direct relationship between the concentration of
this bio-catalyst solution and the fast capability to degrade the organic
matter present in a contaminated environment.
The effect of the bio-catalyst on the bioremediation process was also
assessed in the absence of the pollutants (crude oil hydrocarbons).

From the beginning of the test, the sample labeled D presented a
rapidly decreasing of COD values. This strengthens the premise that
this product can amend bioremediation conditions, speed up bio-
restoration rates, and improve Oxygen transfer to the water. These
properties allowed this sample to end the experimentation with a
%RCOD of 86.949 ± 0.125.

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4. Conclusion
The effectiveness of a commercial bio-catalytic agent used on
bioremediation of Total Petroleum Hydrocarbon-contaminated
seawater was evaluated in this study. The analyzed bio-catalyst
showed positive outcomes for the drop-collapse test and oil-spreading
assay. The measurement of emulsification activity (E 24 ) and Critical
Micelle Concentration (CMC) displayed values of 74.47% and 40 mg
L −1 , respectively. All these values were higher than the satisfactory
values reported in literature confirming the good quality of the
product.
The quality values obtained were corroborated through the study of
the degradation ability of the bio-catalyst. It enhanced the remediation
process to different extents. The decrement of COD, turbidity, and DO
content was noticeable in the crude oil-contaminated samples with
bio-catalytic agent solution added and subjected to aeration systems.
The highest TPH removal efficiency was reached by the sample
labeled F, which contained 640 mg L −1  of petroleum and 10000 mg
L −1  of bio-catalyst solution. The significant reduction of 81.537%
allowed us to recognize this sample as the best water quality of the
analyzed samples.
Furthermore, it was determined that agitation and aeration systems
have an essential effect on the bioremediation process. As a matter of
that, TPH removal efficiencies for aerated samples were in a range of
70%–82%. At the same time, those not subjected to an agitation
system achieved only a near value of 20%. Unequivocally, catabolic
cooperation between groups of microorganisms is important during
the bioremediation process, because sometimes the complete

petroleum hydrocarbons degradation by an only microorganism is not
possible. This buttresses the preference of many investigators to apply
the bio-augmentation method, or the bio-stimulation and bio-
augmentation simultaneously with a consortium of microbial strains
belong to different genera to attain best water quality and optimal
results during remediation of crude oil-contaminated sites.