International Journal of Environmental Science and Technology (2022) 19:2823–2834

https://doi.org/10.1007/s13762-021-03325-y

ORIGINAL PAPER

Comparative evaluation of different bioremediation techniques

for crude oil‑contaminated soil
M. G. Silva1 · L. M. Volcão2,3 · E. R. Seus1 · M. I. Machado1 · N. Mirlean1 · P. R. M. Baisch1 · F. M. R. da Silva Júnior2,3 

Received: 26 October 2020 / Revised: 22 March 2021 / Accepted: 13 April 2021 / Published online: 22 April 2021
© Islamic Azad University (IAU) 2021

Abstract
The present study aimed to establish, using the bioremediation technique, the most efficient treatment strategy for remediation
of a sandy soil that is artificially contaminated with light crude oil. The experiment was conducted for 180 days to monitor
the biodegradation of oils and greases (OG’s), aliphatic hydrocarbons (AHs), and polyaromatic hydrocarbons (PAHs). The
removal of these compounds was evaluated using three different strategies, the addition of nutrients (III), addition of a bio-
surfactant produced from Pseudomonas sp. (IV), and the addition of the biosurfactant plus nutrients (V). The remediation
strategies were compared with the control containing only soil (I) and the control with natural crude oil attenuation (II). At the
end of the study, it was observed that the strategies III and V showed the best OG’s removal rates, i.e., 90.40% and 78.00%,
the best AHs removal rates, i.e., 96.82% and 98.35%, and best PAH removal rates, i.e., 83.58% and 72.3% respectively.
Based on the above results, it was concluded that the bioestimulation methods are important tools in the remediation of the
environment impacted by oil spills. In addition, the study suggests that the knowledge of the native microbiota is important
to improve the efficiency of these bioestimulation techniques.

Keywords  Petroleum · Oil spill · Biosurfactant · Nutrients · Hydrocarbons

Introduction Crude oil spills can occur at different stages of petro-

At the present, environmental pollution is one of the most
complex problems, primarily caused by increased industri-
alization and continuous population growth. The new era has
resulted in extreme dependence on petrochemical products,
making fossil fuels major contributors to environmental con-
tamination (Koshlaf and Ball 2017).
Editorial responsibility: Josef Trögl.
* F. M. R. da Silva Júnior
f.m.r.silvajunior@gmail.com
1
Laboratório de Oceanografia Geológica, Instituto de
Oceanografia, Universidade Federal do Rio Grande, Avenida
Itália, km 8, 96203‑900 Rio Grande, Brasil
2 The ecological potential and low cost are some of the
Programa de Pós‑Graduação em Ciências Da Saúde,
Faculdade de Medicina, Universidade Federal do Rio Grande,
Rua Visconde de Paranaguá, 102, 96203‑900 Rio Grande,
Brasil
3
Laboratório de Ensaios Farmacológicos e Toxicológicos,
Instituto de Ciências Biológicas, Universidade Federal
do Rio Grande, Avenida Itália, km 8, 96203‑900 Rio Grande,
Brasil
leum processing, such as pumping, transport, and refining.
After the spill, crude oil is subjected to processes such as
spreading, evaporation, dissolution, photodegradation, and
biodegradation (Stiver and Mackay 1984; Boopathy et al.
2012; Radović et al. 2014). All these processes influence
the choice of appropriate measure for the remediation of
the impacted environment. The remediation of the affected
environment is becoming increasingly difficult because the
contaminants can persist for a longer period. The effect of
crude oil hydrocarbons on the ecosystem, and their impact
on environmental and human health, is currently the focus of
the research in this area. Soil pollution represents a serious
and widespread environmental risk, attracting public and
scientific attention (Da Silva Júnior et al. 2012, 2013; Kosh-
laf and Ball 2017; De Almeida et al. 2018).
benefits of bioremediation compared to both chemical and
physical methods of remediation (Azubuike et al. 2016).
Although bioremediation is an incomplete hydrocarbon
catabolism strategy that is time-consuming (Da Silva Júnior
et al. 2014), it is the most environmentally friendly reme-
diation approach, as it does not require expensive chemical,

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2824
mechanical, and intensive interventions (Truskewycz et al.
2019). Bioremediation can be performed through bio-aug-
mentation, where exogenous microorganisms are added to
the contaminated environment, as the native microbiota is
often not adapted and is not able to degrade the a variety
of contaminants (Barry et al. 1997; Da Silva Júnior et al.
2018). In bioestimulation, nutrients containing nitrogen (N)
and phosphorus (P) that are essential to the microorganisms
and responsible for the degradation of contamination must
be added (Hoff 1993).
Bioremediation techniques can suffer from some limita-
tions, as microorganisms often do not have access to pollut-
ing molecules (hydrocarbons) that are attached to the soil
matrix. Thus, it is necessary to make this pollutant fractions
bioavailable. In such a scenario, the use of a surfactant can
enhance the action of microorganism against the pollutants,
thus promoting a higher pollutant degradation rate (Yan
et al. 2016; Ebadi et al. 2017). There are several studies
in the literature that apply biosurfactants; however, there is
a non-uniformity in the development of technology, espe-
cially in developing countries. These countries, such as Bra-
zil, have enormous diversity of soil morphotypes, as well
as peculiar physical–chemical conditions compared to soils
in developed countries located in the northern hemisphere.
This scenario puts the soils of these regions at risk, which
already lack environmental legislation and inspection. Thus,
studies with specific conditions can contribute to the under-
standing and solution of these problems.
Fig. 1  Sampling point of the soil used in this study, located in Rio Grande, Rio Grande do Sul, Brazil
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International Journal of Environmental Science and Technology (2022) 19:2823–2834
Added to this, there is a discussion on which of the two
techniques, bio-augmentation or bioestimulation, is a better
strategy for bioremediation. While some authors demon-
strate that selectivity and specialization of added microor-
ganisms mainly defines the bioremediation efficacy, others
argue that bioestimulation can provide suitable nutrients and
conditions for both indigenous and exogenous microbes. Due
to limitations associated with each technique when applied
individually, these are emerged as complementary (Tyagi
et al. 2010). In this paper, we optimize nutrients addition
in a joint bioestimulation and bio-augmentation approach
to cleanup soil contaminated with crude oil, observing the
efficiency of a biosurfactant produced by a microorganism
isolated from the Rio Grande, Rio Grande do Sul, Brazil,
for bioremediation in the presence and absence of nutrients.
Materials and methods
Crude oil and soil collection
Ipiranga Refinery supplied the crude oil used in this study.
The oil supplied was light crude oil of the Hydra type with
and API 49.3° and a density of 0.778 g/cm3. The soil used in
this study was collected along the Lagoa dos Patos estuary
in the Rio Grande, Rio Grande do Sul, Brazil (32° 01′ 40" S,
52° 05′ 40" W) (Fig. 1). The collection point is located near
International Journal of Environmental Science and Technology (2022) 19:2823–2834 2825
the pipeline that links the Transpetro (TERIG) terminal to
the Ipiranga Refinery.
Biosurfactant production
The biosurfactant was produced by a bacterial strain which
was previously isolated from the Rio Grande region in the
Laboratório de Engenharia de Bioprocessos (Universidade
Federal do Rio Grande) and classified by the Laboratório de
Microbiologia as Pseudomonas aeruginosa LBM10 (Quines
et al. 2004).
The strain was activated in tryptic soy agar (TSA) tubes
and then cultivated at 30 °C for 48 h. The rhamnolipid bio-
surfactant production was carried out by fermentation in an
Erlenmeyer flask containing synthetic medium as follow:
­NH4NO3 (0.05 M), ­KH2PO4 (0.03 M), ­Na2HPO4 (0.04 M),
­MgSO4 (8.0 × ­10–4 M), ­CaCl2 (7.0 × ­10–6 M), ­Na2EDTA
(4.0 × ­10–6 M), and soybean oil (40 g/L). The bacterial inoc-
ulum was scraped with peptone water (0.1%) and added to
the Erlenmeyer flask. The flask was then placed in a rotary
incubator at 30 °C and 180 rpm for 48 h. Further, the flask
content was centrifuged at 6,000 rpm for 15 min, and the
precipitate was discarded (Pietro et al. 2008).
The supernatant product was used to determinate the
emulsification index ­(E24) and the biosurfactant glycerol
concentration, the pellet was used to quantify the biomass.
The E24 of the culture samples was calculated according
Bicca et al. (1999), through the percentage of the height
of the emulsified layer (mm) divided by the total height of
the liquid column (mm). Control assays were performed
using an unfermented medium instead of the supernatant.
The rhamnolipid biosurfactant concentration was expressed
as rhamnose (g/L) and measured using the phenol–sulfuric
method (Dubois et al. 1956). The absorbance was measured
at 420 nm in a spectrophotometer (Biospectro SP-22, China)
with rhamnose as the standard. The biomass was moni-
tored according Wu and Ju (1998), in a calibration curve of
between OD600, and the cell dry-weight concentration (g/L)
was first established.
Experimental design
The crude oil spill was simulated in glass boxes containing
10 kg of soil such that the proportion of oil was 4% w/w
soil. Further, three different bioremediation strategies were
adopted, similar to Zilio et al. (2017), namely the addition
of nutrients (III), the addition of biosurfactant (IV), and the
addition of biosurfactant plus nutrients (V). The experiment
was carried out in three replicates for each treatment. Dif-
ferent bioremediation strategies were compared with two
controls, i.e., soil control containing water (I) and control
containing only natural crude oil (II). The nutrients added
to the soil were composed of N in the form of urea (46% N)
and P in the form of simple superphosphate (18% P), at a
C:N:P ratio of 100:1.25:1 (Trindade et al. 2005). The biosur-
factant was added to the soil in a ratio of 1:1 v/v crude oil/
biosurfactant (Bento 2005). The boxes placed at a humidity
of about 65–80% of the field capacity and monitored over six
months according to the method described by Sarkar et al.
(2005). The temperature and pH of the soil were monitored
as previously described (Camargo et al. 1986).
Experimental analysis
Soil sampling
The soil sampling was randomly carried out by introducing
12-cm-long tubes into the soil at approximately 5 cm depth.
The collected samples were homogenized and quartered. A
part of the sample was packed in sterile bottles for micro-
biological analyzes that were processed on the same day.
The other part of the sample was homogenized and dried
at room temperature (± 25 °C), disaggregated, and stored
in glass flasks for physical and chemical analysis (Table 1).
Nutrient analysis
Total organic carbon (TOC) was determined by the method
given in Strickland and Parsons (Nie et al. 1970) with modi-
fications. Total N was determined using the micro-Kjeldahl
method (Tedesco et al. 1995). During the analysis of total P,
the soil samples were calcined to eliminate organic matter
and treated with a hydrochloric acid solution (Ruttenberg
1992). P was determined by the addition of ascorbic acid
and ammonium molybdate to the sample. A blue-colored
phosphomolybdate complex was formed, which was meas-
ured by the colorimetry reaction in a spectrophotometer at
λ = 885 nm.
Monitoring of the microbial population
The microbiota was monitored by conducting a mesophilic
bacterial count. The agar plates were inoculated with the
sample from each flask and incubated for 48 h at 36 ± 1 °C.
After incubation, the plates containing between 25–250
Table 1  Physical and chemical characteristics of the soil used in the
experiment
Parameter Measurement Parameter Measurement
Sand (%) 96.8 Nitrogen (%) 0.02 ± 0.001
Silt (%) 2.3 Phosphorus (%) 0.10 ± 0.006
Clay (%) 0.9 Organic Carbon (%) 0.27 ± 0.018
Porosity 0.65 Oils/greases (%) 0.05
pH 7.88 Density (g/cm3) 1.23
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2826 International Journal of Environmental Science and Technology (2022) 19:2823–2834
colonies were counted to determine the mesophilic bacte-
rial count (APHA 2005).
Determination of oil and grease
Oil and grease (OG) content was determined according to
the gravimetric method described by Rizzo and Raimundo
(Trindade et al. 2005), using the ultrasound technique to
extract hydrocarbons. The extract was transferred to a previ-
ously weighed flask and concentrated on a rotary evaporator
using nitrogen gas. The flask was weighed again, and the
difference between the initial and final weights of the flasks
was used to determinate the OG content in the samples.
Hydrocarbon extraction and analysis
The hydrocarbons were analyzed following the recommen-
dations provided in “Reference methods for marine pollution
studies, No. 20: determination of crude oil hydrocarbons
in sediments” (UNEP 1992). For extraction, initially, the
soil samples were dried at room temperature, disaggregated,
homogenized, and weighed. Further, surrogate standards
(naphthalene-D-8, phenanthrene-D-10, crizene-D-12, per-
ylene-D-12 eicosene, and tetradecene) were added to the
soil samples. The soil samples were then extracted using a
pesticide-grade solvent mixture (50% dichloromethane and
50% n-hexane) in a Soxhlet type extractor for 12 h. Cop-
per blades, cleaned and activated with the hydrochloric acid
solution, were used to eliminate possible contamination
by sulfur compounds during extraction. The soil extracts
were concentrated on a rotary evaporator to approximately
3 mL. In addition, a small amount of sodium sulfate was
also added to ensure complete removal of water. Further,
the concentrated extracts were subjected to adsorption chro-
matography in a glass column (clean up) to separate the
fraction of aliphatic hydrocarbons (AHs) from the aromatic
fraction. The AHs (C12 to C36) were determined on a Perki-
nElmer Clarus 500 gas chromatography coupled to a flame
ionization detector (GC-FID) using the Elite-1 chromato-
graphic column (Crossbond dimethyl polysiloxane 100%,
30 m × 0.25 mm I.D., df = 0.25 μm) and automatic sample
injector. The polyaromatics hydrocarbons (PAHs) were ana-
lyzed on a PerkinElmer Clarus 500 gas chromatography cou-
pled to mass spectrometry (GC–MS) using Elite-5MS col-
umn (diphenyl 5%–dimethylsiloxane 95%, 30 m, 0.25 mm
I.D., df = 0.25  μm) and automatic sample injector. The
analysis was conducted using the selected ion monitoring
mode, during which the characteristic ions corresponding to
the mass/charge (m/z) of fragments of each compound were
selected and monitored. The recovery of the methodology
for aliphatic hydrocarbons was assessed using the 1-eicosene
standard as described in U.S. EPA 8015C (U.S. EPA 2013).
For polyaromatic hydrocarbons, the recovery of the method
13
was evaluated using the surrogate standard p-terfenil-d14,
as described in U.S EPA 8270E (U.S. EPA 2014). The CRM
47,543 standard was used, along with other HPA standards
provided by the company Sigma–Aldrich: 2,6-Dimethyl-
naphthalene (2,6DMN), Biphenyl (BIF), Dibenzothiophene
(DBZT), Benzo (e) pyrene, and perylene.
Statistical analysis
Nutrient data (TOC, N, and P) and removal rate were ana-
lyzed using Excel® and Statistica® software (ANOVA fol-
lowed by the Tukey’s test) at a 5% significance level. The
assumptions of the normality of the data were tested using
the Kolmogorov–Smirnov test.
Results and discussion
Soil and biosurfactant characterization
The physicochemical properties of the soil collected for the
experiment were previously described. The soil is classi-
fied as sandy soil and allows good diffusion of oxygen, thus
facilitating the aerobic degradation process. It has be noted
that the sample collection region is strongly influenced by
the anthropogenic supply of nutrients from the industrial
district of the Rio Grande city.
The concentration obtained from the rhamnolipid bio-
surfactant was 1.24 g/L (­ E24 = 62%) in 48 h cultivation. P.
aeruginosa is capable of producing different types of rami-
nolipids with similar chemical structure and surfactant activ-
ity (Gogoi et al. 2003), being among the most effective and
applied in several bioremediation processes (Kumari et al.
2012; Ebadi et al. 2017).
Nutrient analysis
The variational trend in the concentration for nutrients (N,
P, and TOC) is shown in Fig. 2. In can be observed that val-
ues are more or less constant in control I. Significant differ-
ences between the initial and final N (Fig. 2a) and P (Fig. 2b)
levels were observed only in the bioremediation strategies
involving nutrients addition. However, there were signifi-
cant variations in TOC in all the strategies (III–V) involving
crude oil addition and control, according to Tukey’s test. The
difference was most pronounced in III, with a reduction of
approximately 70% of the initial value, while other treatment
strategies showed a reduction of 27–53%.
Although crude oil is a carbon source for microorgan-
isms, it does not provide other nutrients necessary for the
cell growth and metabolic activities (Marchand et al. 2017).
In the natural environment, hydrogen and oxygen are
supplied by water, whereas N and P are usually found in
International Journal of Environmental Science and Technology (2022) 19:2823–2834 2827
Fig. 2  Values (%) of nitrogen (a), phosphorus (b), and carbon (c) dur-
ing 180  days, in the different strategies, (I) soil control, (II) natural
crude oil, (III) addition of nutrients, (IV) addition of biosurfactant,
and (V) addition of biosurfactant plus nutrient
insufficient quantities. Thus, nutrients present in the environ-
ment in smaller quantities are limiting factors for the natural
biodegradation of hydrocarbons. Generally, one or both of
these compounds should be added to the soil for effective
bioremediation (Asquith et al. 2012; Mejeha et al. 2019).
Bacterial counts
The logarithmic of bacterial growth in CFU/g during the
180 days is shown in Fig. 3. As shown in Fig. 3, the expo-
nential growth in CFU observed during the first few days
of the study indicated the multiplication of fast-growing
bacteria. On the 30th day, the bacterial population stabi-
lized and started to reduce due to the probable decrease in
Fig. 3  Logarithm of bacterial growth in Colony Forming Units per
gram of soil (­Log9 CFU/g) in the different strategies, (I) soil control,
(II) natural crude oil, (III) addition of nutrients, (IV) addition of bio-
surfactant, and (V) addition of biosurfactant plus nutrients, during
180 days
nutrients. Added to this, the hydrocarbon bioremediation
can generate intermediate compounds, which can be toxic
to microorganisms (Łebkowska et al. 2011). It should also
be noted that experimental conditions in the present study
such as pH close to neutral, temperature of 25 ± 5 °C, soil
aeration, presence of essential nutrients, and humidity of
65–80% are beneficial for effective bacterial biodegradation
(Najirad et al. 2012; Liu et al. 2017).
Further, it was observed that I, II, and III presented a
lower number bacterial count when compared with the strat-
egies involving biosurfactant addition (i.e., IV and V). This
fact is probably due to the presence of P. aeruginosa IV and
V since the biosurfactant was not sterilized. The use of bac-
teria native to the contaminated environment for the produc-
tion of biosurfactants becomes an advantage in relation to
the use of biosurfactants produced with bacteria exogenous
to the environment. In addition, native microorganisms are
already adapted to the environment, making the bioremedia-
tion process more efficient (Zhuang et al. 2002). Added to
this, it was possible to observe that the biosurfactant used
did not cause inhibition of the local microbial population in
comparison with the natural attenuation.
Oil and grease removal
Crude oil and its derivatives are the major environmental
pollutants (da Cunha et al. 2012), because they can be pre-
sent in the environmental for a longer period when they
adhere to the soil particles. As shown in Fig. 4, there was
an effective reduction in the OG content in II during the
first month. However, this reduction was stabilized in the
subsequent months. This represents natural crude oil weath-
ering subjected to a large number of natural processes, such
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2828 International Journal of Environmental Science and Technology (2022) 19:2823–2834
Fig. 4  Percentage of oil and grease remaining in (I) soil control, (II)
natural crude oil, (III) addition of nutrients, (IV) addition of bio-
surfactant, and (V) addition of biosurfactant plus nutrients, during
180 days
as volatilization and biodegradation (Boopathy et al. 2012;
Radović et al. 2014; Kim et al. 2020).
In treatment strategies involving the addition of nutrients,
biosurfactant, or both, greater efficiency in the removal of
these compounds was observed. In III, involving only the
addition of nutrients, a higher percentage of OG removal
(90.4%) was observed. Abdulsalam et al. (2012) evaluated
the bioremediation of a soil contaminated with used motor
oil under six different combinations of treatments, including
addition of NPK (20:10:10) and exogenous bacteria. They
observed a greater OG removal in the first few weeks of the
experiments and demonstrated that the strategy involving
only the addition of nutrients (nitrogen, phosphorus, and
potassium) and water was more efficient in OG removal, i.e.,
75% of the initial values.
Biodegradation of petroleum hydrocarbons
In the present study, generally, the n-alkanes (C12-C36) of
AHs and the isoprenoid alkanes (pristine and phytane) were
observed (Supplementary data S1). Heavy AHs (> C20)
were not observed in I, while the lighter fraction of AH was
observed, and its concentrations varied over the experimen-
tal period. It should be noted that the soil used in the experi-
ments was collected from a region close to the industrial
district of Rio Grande as well as close to a road. Thus, the
soil may be exposed to various types of contaminants, which
may explain the detection of certain hydrocarbons in this
soil.
The removal percentage of AHs in II, IV, and V at the
end of the experimental period of 180 days are given in
Table  2. The highest AH removal was observed when
the nutrients were added, i.e., 92.82% in III (addition of
nutrients) and 98.35% in V (biosurfactant + addition of
13
Table 2  Removal (%) of aliphatic hydrocarbons in treatments with
addition of nutrients (III), biosurfactant (IV), and addition of biosur-
factant plus nutrients (V)
Treatment Days Aliphatic (µg/g) Removal (%)
III 0 229.39 ± 25.2 92.82a
90 149.75 ± 10.5
180 7.29 ± 0.36
IV 0 1418.48 ± 170.2 55.40b
90 658.24 ± 69.1
180 632.68 ± 50.6
V 0 1492.80 ± 132.9 98.35a
90 67.55 ± 8.1
180 24.65 ± 2.4
In evaluating the differences between the removal rates, different let-
ters indicate statistical difference between treatments, considering the
value of p < 0.05
nutrients). Ebadi et al. (2017) demonstrated that biosur-
factant-producing P. aeruginosa was able to degrade crude
oil, even in the presence of contaminated saline soil. They
also observed that the inoculation of a consortium with
strains of this species led to significant degradation of
crude oil hydrocarbons.
The GC chromatogram revealed an effective removal
of AHs, including heavier fractions in III (Fig. 5) and V
(Fig. 6), compared to treatments II (Fig. 7) and IV (Fig. 8)
that did not achieve the same efficiency in removal of ali-
phatic hydrocarbons. Kumari et al. (2012) observed degra-
dation of n-alkanes up to 80% using the biosurfactant pro-
duced by Pseudomonas sp. They also note concluded that
the reduction in the surface tension of the medium, after
biosurfactant addition, was partly responsible for the deg-
radation efficiency of crude oil, as it increases the bioavail-
ability of the hydrophobic substrate for the bacterial cells.
Despite not being considered as a highly toxic group,
AHs are important indicators of petrogenic contamination.
On the other hand, PAHs are organic pollutants of great
environmental concern and toxicological interest because
of their carcinogenic and mutagenic potencies across species
(Bhatia et al. 2018). In addition to lighter AHs, lighter PAHs
(2 and 3 rings), phenathrene and chrysene were identified
in I. However, at the end of the 180 days, only chrysene
was detected (Supplementary data S2). A more effective
reduction of lighter hydrocarbons when compared to that
of heavier hydrocarbons was observed during the natural
degradation of crude oil in II. The process of natural attenu-
ation of an organic pollutant in the soil, i.e., without the
addition of any nutrients or without the addition of microbes
adapted to the contaminated environmental conditions, can
occur continuously because of the natural adaptation process
of the native soil microbiota (Declercq et al. 2012; García-
Sánchez et al. 2018).
International Journal of Environmental Science and Technology (2022) 19:2823–2834 2829

Fig. 5  Chromatographic analy-
sis for aliphatic hydrocarbons
in strategies with addition of
nutrients (III) at time 0 (a) and
180 days (b)

Fig. 6  Chromatographic analy-
sis for aliphatic hydrocarbons
in treatment with addition of
biosurfactant plus nutrients (V)
at time 0 (a) and 180 days (b)

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2830 International Journal of Environmental Science and Technology (2022) 19:2823–2834
Fig. 7  Chromatographic analy-
sis for aliphatic hydrocarbons in
strategies with natural crude oil
(II) at time 0 (a) and 180 days
(b)
The reduction in PAHs was observed in II, IV, and V, as
shown in Table 3. The most effective removal of PAHs was
observed in III involving only the nutrients addition (i.e.,
95.75% and 71.55% for the heavier and lighter hydrocar-
bons, respectively). Grace Liu et al. (2011) showed that bio-
augmentation methods were more effective in the removal
of polar aromatic fractions from crude oil, with the addition
of a higher C:N:P ratio than that used in the present study
(100:11:3.7). However, it should be noted that the nutrients
should be added with caution, depending on the environmen-
tal conditions. Zeneli et al. (2019) observed a lower PAH
removal than that reported in the present study while carry-
ing out bioestimulation at C:N:P ratio of 100:10:1.
Although V (i.e., in the presence of biosurfactant and
nutrients) does not result in the efficient removal of heav-
ier aromatic fractions, it leads to the effective removal of
lighter PAHs (Table 3). It should be noted that the bioavail-
ability of hydrocarbons and nutrients to the microbial bio-
mass is a crucial factor determining the biodegradation of
high-molecular-weight PAHs (Xu et al. 2005). Our study
demonstrated similar degradation efficiencies to those
obtained by Kumari et al. (2012) during the degradation
of certain PAHs, such as phenanthrene (78.62%), fluoran-
thene (60.76%), and pyrene (56.52%) (Supplementary data
13
S2). They also observed a significant reduction in the sur-
face tension as a result of the biosurfactant production by
Pseudomonas sp. BP10. The biosurfactant can facilitate
the adhesion of hydrocarbons from crude oil to the bacte-
rial cells, making it bioavailable.
Some studies using other species of microorganisms have
shown that biosurfactants, by causing the dispersion of the
oil, could stimulate the subsequent biodegradation of the
spilled contaminant through the action of natural microor-
ganisms to the environment (Saeki et al. 2009; Durval et al.
2018). Cai et al. (2016) when developing hyper produc-
ing mutants that produced biosurfactants as oil dispersion
agents, from a Rhodococcus erythropolis strain isolated from
oily wastewater, observed an increase in productivity and the
corresponding dispersion efficiency when compared to the
biosurfactants produced by the wild type strain.
Bioremediation studies involve the search for satisfactory
models for the recovery of degraded sites and obviously have
a close relationship with practical application. It is also clear
that the results found in laboratory studies, in micro- and
mesocosms and semi-fields, cannot be extrapolated directly
to bulk operations. As mentioned earlier, environmental and
economic conditions are important for the choice of models
International Journal of Environmental Science and Technology (2022) 19:2823–2834 2831

Fig. 8  Chromatographic analy-
sis for aliphatic hydrocarbons
in strategies with addition of
a biosurfactant produced from
Pseudomonas sp. (IV) at time 0
(a) and 180 days (b)

Table 3  Removal (%) of Treatment Days Σ 2–3 rings (µg/g) Removal (%) Σ 4–6 rings (µg/g) Removal (%)
polyaromatics hydrocarbons
in treatments with addition of III 0 3.038 ± 0.2 95.75a 0.639 ± 0.05 71.55c
nutrients (III), biosurfactant
90 1.343 ± 0.1 0.420 ± 0.03
(IV), and addition of
biosurfactant plus nutrients (V) 180 0.129 ± 0.01 0.182 ± 0.01
IV 0 2.383 ± 0.2 81.91b 1.351 ± 0.1 41.78d
90 0.148 ± 0.02 1.241 ± 0.1
180 0.431 ± 0.03 0.786 ± 0.06
V 0 7.057 ± 0.3 94.74a 1.202 ± 0.1 49.87d
90 2.741 ± 0.2 1.024 ± 0.1
180 0.371 ± 0.03 0.603 ± 0.08

In evaluating the differences between the removal rates, different letters indicate statistical difference
between treatments, considering the value of p < 0.05

13

2832
that must be implemented and this means that different mod-
els need to be developed to meet local specificities.
Monitoring the progress of oil bioremediation processes
in contaminated soils, such as those carried out in the pre-
sent study, is essential for the diversification of the database
that feed studies that apply mathematical models applied to
evaluate the efficiency of bioremediation alternatives (Sanni
and Emetere 2016; Ojewumi et al. 2017). In addition, the
aforementioned models are of paramount importance in
helping to conduct the use of the large-scale bioremediation
process, helping to conduct its application in real scenarios.
We emphasize that this study evaluated the hydro-
carbon degradation occurred over a wide period of time
(180 days). Shorter times than those used in the present
study may not be sufficient for an evaluation of biologi-
cal variables, such as reducing toxicity and restoring less
disturbed communities (Molina-Barahona et  al. 2005;
Hubálek et al. 2007; Shen et al. 2016; Da Silva Júnior
et al. 2014). Advances in the area are essential, since the
implementation of bioremediation techniques depends on
local characteristics, with environmental conditions, time,
space, and local legal restrictions (Cristorean et al. 2016).
This takes on more important dimensions in countries like
Brazil, where the number of contaminated sites has not
been measured and where legal devices are extremely frag-
ile (Da Silva Júnior 2020).
Conclusion
The results obtained in this study showed that bioestimula-
tion methods are important tools in remediating the envi-
ronment impacted by crude oil. Among the bioremediation
strategies applied, the one involving the addition of nutri-
ents (III) and the addition of biosurfactant plus nutrients
(V) showed the best efficiency in oil and greases removal,
in addition to the removal of aliphatic hydrocarbons and
aromatic fractions. This study also pointed out that differ-
ent bioremediation strategies enable the growth of differ-
ent bacterial strains, with peculiar degradation character-
istics, thus suggesting that future studies must be focused
on the isolation of promising degrading microbial species
for crude oil contaminated environment.
Supplementary Information  The online version contains supplemen-
tary material available at https://​doi.​org/​10.​1007/​s13762-​021-​03325-y.
Acknowledgements  This study was financed in part by the Coorde-
nação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil
(CAPES)—Finance Code 001. Human Resources Program of the
National Crude oil Agency—PRH—27/MCT/ANP—supported in the
development of this research.
13
International Journal of Environmental Science and Technology (2022) 19:2823–2834
Declarations 
Ethical approval  This article does not contain any studies with human
participants or animals performed by any of the authors.
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