Biodegradation

https://doi.org/10.1007/s10532-021-09944-z (0123456789().,-volV)
(0123456789().,-volV)

ORIGINAL PAPER

Bacterial biosurfactant increases ex situ biodiesel

bioremediation in clayey soil
Andressa Decesaro . Alan Rempel . Thaı́s Strieder Machado . Ângela Carolina Cappellaro .
Bruna Strieder Machado . Iziquiel Cechin . Antônio Thomé . Luciane Maria Colla

Received: 19 September 2020 / Accepted: 12 April 2021

Ó The Author(s), under exclusive licence to Springer Nature B.V. 2021

Abstract The contamination of soils by oily com-
pounds has several environmental impacts, which can
be reversed through bioremediation, using biosurfac-
tants as auxiliaries in the biodegradation process. In
this study, we aimed to perform ex situ bioremediation
of biodiesel-contaminated soil using biosurfactants
produced by Bacillus methylotrophicus. A crude
biosurfactant was produced in a whey-based culture
medium supplemented with nutrients and was later
added to biodiesel-contaminated clayey soil. The
Supplementary Information The online version contains
supplementary material available at https://doi.org/10.1007/
s10532-021-09944-z.
produced lipopeptide biosurfactant could reduce the
surface tension of the fermentation broth to 30.2 mN/
m. An increase in the microbial population was
observed in the contaminated soil; this finding can
be corroborated by the finding of increased CO2
release over days of bioremediation. Compared with
natural attenuation, the addition of a lower concentra-
tion of the biosurfactant (0.5% w/w in relation to the
mass of diesel oil) to the soil increased biodiesel
removal by about 16% after 90 days. The added
biosurfactant did not affect the retention of the
contaminant in the soil, which is an important factor

A. Decesaro  A. Rempel  T. S. Machado  Â. C. Cappellaro

A. Thomé  L. M. Colla (&) Environmental Engineering Course, Faculty of
Civil and Environmental Engineering, Faculty of Engineering and Architecture, University of Passo Fundo,
Engineering and Architecture, University of Passo Fundo, BR 285, km 171, Passo Fundo, RS, Brazil
Campus I, Building L1, BR 285, km 171, Neighborhood e-mail: angelzir@hotmail.com
São José, Mailbox 611, Passo Fundo,
RS CEP: 99052-900, Brazil B. S. Machado
e-mail: lmcolla@upf.br Chemical Engineering Course, Faculty of Engineering
and Architecture, University of Passo Fundo, BR 285, km
A. Decesaro
171, Passo Fundo, RS, Brazil
e-mail: andressa_decesaro@hotmail.com
e-mail: 173882@upf.br
A. Rempel
e-mail: alan.rempel@hotmail.com I. Cechin
Environmental Engineering Course, Faculty of
T. S. Machado
Engineering and Architecture, University of Passo Fundo,
e-mail: thaiis.strieder@hotmail.com
BR 285, km 171, Passo Fundo, RS, Brazil
A. Thomé e-mail: iziquielcecchin@upf.br
e-mail: thome@upf.br

123

to be considered when applying in situ bioremediation
technologies.
Keywords Oily contaminants  Bacillus
methylotrophicus  Lipopeptide  Contaminant
retention
Introduction
Soil contamination is a growing global concern
because it causes an environmental imbalance in the
ecosystem, thereby hampering the environment and
public health (Gupta 2020; Mao et al. 2015). This
contamination is mainly caused by the irregular
disposal of industrial and mining wastes, in addition
to environmental accidents related to the transporta-
tion of fuels and dangerous substances (Martinez-
Costa and Leyva-Ramos 2017; Pacwa-Płociniczak
et al. 2011). Fuel spills cause a series of changes in
soil, water, air, fauna, and flora quality (Wang et al.
2019; Mcwatters et al. 2016; Snape et al. 2006).
Therefore, the impacted areas should be treated on
priority through recovery processes using various
physical, chemical, and biological techniques (Khal-
ladi et al. 2009).
Bioremediation is a possible option for the recovery
of areas contaminated with oily compounds. It is more
sustainable than physicochemical processes (Gupta
2020; Vidonish et al. 2016). In the bioremediation
process, contaminants, such as biodiesel, are used as
sources of carbon and energy by microorganisms
(Gupta 2020; Yalaoui-Guellal et al. 2020). However,
characteristics of the soil and the physicochemical
properties of these oily contaminants affect their
bioavailability in the environment. In addition, due to
the high lipophilicity of the contaminant, there is a
limited interaction between biodiesel and microor-
ganisms due to the difficulty in crossing cell mem-
branes, which causes the contaminant to remain in
contact with soil particles for a long period of time,
being this the driving force to start the sorption
process. Sorption is responsible for the ‘‘trapping’’ of
contaminants, removing them from the dissolved
state. Consequently, if the compounds are heavily
adsorbed, they may be unavailable to microorganisms,
thus making difficult their biodegradation (Gupta
123
Biodegradation
2020; Oualha et al. 2019; Chaprão 2015; Alexander
2000).
Microorganisms bypass this obstacle by excreting
biosurfactants into the soil; this increases the surface
area of the contaminant and enables the entry of
nonpolar molecules into the cell, thereby facilitating
microbial access (Machado et al. 2020; Pereira et al.
2019; Mao et al. 2015; Tonini et al. 2010; Fontes et al.
2008). However, the mechanisms underlying the
effect of biosurfactants on biodiesel retention in
clayey soils remain unknown.
The search for microorganisms that produce bio-
surfactants and oil degraders is being conducted
worldwide. Several microbial genera have already
been listed as biosurfactant producers, e.g., Bacillus,
Pseudomonas, and Corynebacterium (Pereira et al.
2019; Syakti et al. 2019; Wang et al. 2019; Chan-
dankere et al. 2014; Hassanshahian et al. 2012).
The use of biosurfactants in this context is an
efficient and sustainable alternative to accelerate the
biodegradation of oily compounds by increasing their
solubility and making them accessible to microorgan-
isms (Machado et al. 2020; Martı́nez-Toledo and
Rodrı́guez-Vázquez 2013; Paria 2008). In addition,
biosurfactants have high selectivity and specific
activity even under extreme pH and temperature
conditions. Therefore, the use of these biocompounds
appears to be a promising alternative to the use of
chemically synthesized surfactants (Felix et al. 2019;
Anjum et al. 2016). However, the use of biosurfactants
is not widespread because of the high production costs
associated with the raw materials used (Decesaro et al.
2020; Fernandes 2010).
The competitive production of biosurfactants using
fermentation processes requires a systematic and
optimized balance of all components, and controls of
temperature, pH, oxygen, and agitation, in addition to
the use of low-cost raw materials, such as the use of
agroindustrial waste (Decesaro et al. 2020; Louhasa-
kul et al. 2020; Vecino et al. 2017). The use of
agroindustrial wastes as whey can lower the costs of
biosurfactant production to competitive levels, in
addition to reducing the environmental problems
associated with their disposal and the treatment costs.
Moreover, whey has high concentrations of nutrients,
such as lactose, proteins, and salts (Decesaro et al.
2020; Alves et al. 2014; Brião and Tavares, 2007;
Domingues and Teixeira 1999). These nutrients can be
incorporated into fermentative processes as substrates
Biodegradation
for the growth of microorganisms, which conse-
quently release biosurfactants that may be included
in the bioremediation process after removal from the
culture media.
In order to contribute with the understanding of the
action of biosurfactants during bioremediation, we
produced biosurfactant with Bacillus methylotrophi-
cus in a whey-based culture medium using fermenta-
tion processes and evaluated its addition over the
biodegradation of biodiesel in clayey soil. In addition,
the effect of adding the biosurfactant on the retention
of the contaminant in the soil was carried out through
tests with contaminated sterile soil.
Methods
Microorganism and biosurfactant production
The microorganism used in this study was isolated
from diesel oil-contaminated soil (Decesaro et al.
2013); it was identified as B. methylotrophicus
CBMB205 (GenBank EU194897) through phyloge-
netic analysis (Decesaro et al. 2020). The bacteria
were maintained in glasses tubes containing plate
count agar (PCA) at 4 °C. The inoculum was prepared
in PC medium consisting of 5 g/L of tryptone, 2.5 g/L
of yeast extract, and 1 g/L of glucose. In 250 mL
Erlenmeyers flasks, the colonies were inoculated
50 mL of PC medium, followed by incubation in an
orbital shaker (model TE-421, Tecnal, Brazil) at
100 rpm for 48 h at 30 °C.
Initially, the whey passed through pre-treatment,
according to the method adapted by Joshi et al. (2008),
where its pH was adjusted to 4 with HCl (1.0 mol/L),
and then boiled for 10 min. After cooling, all the whey
was filtered and the precipitate was removed by
cotton. The filtered solution had its pH adjusted to 7
with NaOH (1.0 mol/L), later being autoclaved and
used as a culture medium.
The culture medium consisted of 50 mL of pre-
treated whey supplemented with 1% (w/v) ammonium
sulfate (132.13 g/mol, Nuclear, Brazil) as the nitrogen
source; 0.5% (v/v) micronutrient solution, composed
of 0.033 g/L NaBr (102.89 g/mol, Neon, Brazil),
0.20 g/L CuSO4.5H2O (249.68 g/mol, Synth, Brazil),
0.81 g/L MgSO4.7H2O (246.48 g/mol, Synth, Brazil),
and 0.31 g/L ZnSO4.7H2O (287.54 g/mol, Quı́mica
Moderna, Brazil) (adapted from Praveesh et al. 2011);
and 2% (w/v) soybean oil as the inducer. Inoculation
was performed using 2 mL of inoculum with an
optical density of 1.0 at 660 nm measured using a
spectrophotometer. Incubation was performed in an
orbital shaker at 100 rpm, 30 °C for 5 days.
After the fermentation process, the biosurfactants
were precipitated following the method proposed by
Dubey and Juwakar (2001) adapted. The cell-free
broth was acidified to pH 2 with HCl (1.0 mol/L),
followed by cooling at 4 °C for 24 h and centrifuga-
tion at 5000 rpm for 10 min. After precipitation, the
sample was lyophilized (Lyronizer LS 3000, Terroni)
by dehydration under vacuum at - 70 °C until use.
To assess biosurfactant production, the surface
tension of the fermentation medium during biosurfac-
tant production was measured in the absence of cells
according to the Du Noüy ring method (Noüy 1919)
using a tensiometer (model Sigma 702, Biolin Scien-
tific, Sweden).
Soil and contaminant
Clayey soil used was obtained from the experimental
geotechnical campus of the University of Passo
Fundo, southern Brazil (geographical coordinates,
28.226320° S and 52.386473° W). The soil for the
bioremediation assay was collected at a depth of 1.2 m
(horizon B) from an open trench at the site. The soil
samples were collected in a deformed state and were
used for physicochemical characterization. Particle
size analysis, Atterberg limits (liquidity and plastic-
ity), specific mass, suction characteristic curve,
hydraulic conductivity, grain density, and porosity
were assessed in accordance with NBR 7181 (ABNT
1984a), 6459 (ABNT 1984b), 7180 (ABNT 1984c),
6508 (ABNT 1984d), and 6457 (ABNT 1986).
Physicochemical characterization was performed
according to the method proposed by Tedesco et al.
(1992). It involved the assessment of the pH of water;
clay content of soil; concentration of organic matter,
existing macronutrients (N, P, K, S, Ca, and Mg),
existing micronutrients (Mn, Zn, Cu, Fe, Mo, B, and
Cl), and exchangeable cations (Al and Na); potential
acidity (Al ? H); cation exchange capacity (CEC);
base saturation; aluminum saturation; and potassium
saturation.
The soil in this study was pedologically classified as
a humic dystrophic Red Latosol (Streck et al. 2008),
geotechnically identified by the unified classification
123

of soils as CH or high plasticity clay. The geotechnical
and physical characterization of the soil was previ-
ously performed by Cecchin et al. (2016). It has a clay
and silt content of 72% and 15%, respectively. Given
the high maturation process of the soil, it has a
different structure from other clayey soils around the
world, with a permeability close to that of sandy soils
(1.39 9 10–3 cm/s).
The soil has an acidic pH (5.1), a high clay content,
and a low CEC (12.7 cmolc/dm3) (Streck et al. 2008),
which are typical characteristics of soils predomi-
nantly consisting of mineral kaolinite clay, given the
structural conformation.
The contaminant examined in this study was pure
soybean biodiesel (B100) obtained from BSBIOS
Company (Passo Fundo, Brazil). According to
BSBIOS (2016), the main components of B100 are
methyl esters from fatty acids (C16-18 and C18
unsaturated), obtained from 100% vegetable oil
derived from soybeans, which is free of aromatic
compounds and sulfur.
Bioremediation assay
The bioremediation assay was performed in hermet-
ically sealed glass reactors (11 cm diameter, 21 cm
height and useful volume of 1.75 L), containing 300 g
of dry soil contaminated with biodiesel at a concen-
tration of 200 g/kg. The bioremediation was per-
formed with the soil’s autochthonous microorganisms,
and this was revolved every 2 or 3 days for oxygen
reinsertion in the bioreactors. The soil moisture was
corrected to 34% with sterile distilled water and the
assays were carried out at room temperature
(25 °C ± 2 °C).
The biosurfactant was added at different concen-
trations in the bioremediation assays, 0.1%, 0.5% and
1.0% (w/w) calculated in relation to the contaminant
mass (200 g/kg), resulting in concentrations of 0.2, 1.0
and 2.0 g of biosurfactant/kg of soil, respectively. The
definitions of the concentrations of the biosurfactant
were based on a previous study by Martı́nez-Toledo
and Rodrı́guez-Vázquez (2013), in which the concen-
tration of biosurfactant produced in the soil was
assessed. For the application, the biosurfactant was
diluted in sufficient sterile distilled water to restore the
ideal soil moisture (34%). The different treatments
123
Biodegradation
were compared with a natural attenuation assay
(without the addition of the biosurfactant) and a
control assay (without the addition of the contaminant
and biosurfactant).
To verify the effects of the clayey soil on the
biodiesel sorption, parallel assays were carried out
with the same experimental configuration; however,
the soil, the biosurfactant, the distilled water and the
equipment were sterilized in an autoclave for 20 min
at 121 °C in properly covered vessels before the start
of the assay. Thus, the contamination retention
verified in the soil, is only related to the sorption
mechanism, since there is no biodegradation process.
All the assays were performed in duplicate and the
duration was 90 days, which covered the adaptation
and growth phases of microorganisms. The Table of
the experimental design of the bioremediation and
retention assays is shown in Supplementary Material.
Measurements and data analyses
In the bioremediation assay, the soil moisture content
was assessed at the initial time point and at 30, 45, 60,
and 90 days using the following equation, according
to NBR 6457 (ABNT 1986):
ðcapsule þ moistsoilÞ  ðcapsule þ drysoilÞ
Moistureð%Þ ¼  100
ðcapsule þ drysoilÞ  ðcapsuleÞ
ð1Þ
The obtained values were used to calculate con-
taminant removal because the calculation was per-
formed in relation to dry soil.
To evaluate the CO2 release, the Bartha and Pramer
(1965) respirometric method was used, which mea-
sures the CO2 release by capturing the CO2 by alkaline
substance (flasks with NaOH, 0.5 mol/L, were placed
inside the hermetically sealed reactors), to occur the
precipitation in the form of barium carbonate
(BaCO3), by the addition of saturated solution of
barium chloride (BaCl2). The excess sodium hydrox-
ide was then titrated with HCl, allowing the calcula-
tion of CO2 release (Mariani 2005). Based on the
volume of HCl spent during acid base titration with the
turning point of phenolphthalein at pH 8.2 to 10.0, it
was possible to determine the amount of CO2 released
by soil microorganisms at each time using the
stoichiometric procedure, from according to Eq. (2).
Biodegradation
The readings were taking every 2–3 days, and after
each reading a new sample of NaOH was inserted into
the flask.
The CO2 release was only measured in assays in
which the soil used was not sterilized. The CO2 release
was only measured in assays in which the soil used
was not sterilized. In soils sterilized by the absence of
live microorganisms, such reading was not performed.
In contrast, in the assays performed without steriliza-
tion, blank sample analyses were performed to con-
sider CO2 capture with a flask without the presence of
soil, in order to consider the possible entry of other
sources of CO2 present in the environment and reduce
the error. The result obtained was inserted into the
equation, through variable ‘‘B’’.
CCO2 ðmgÞ ¼ ðB  V Þ  M  f  6  ðV1  V2 Þ
ð2Þ
where CCO2: carbon of the carbon dioxide, B: HCl
volume spent in the titration of the blank sample, in
other words, without the presence of soil (mL), V: HCl
volume spent on sample (mol/L), M: actual HCl
concentration (mol/L), 6: atomic mass of C (12)
divided by the number of moles of CO2 reacting with
NaOH, V1: NaOH volume used to capture CO2 (mL),
V2: NaOH volume used during titration (mL), and f:
HCl correction factor, average value 0.98.
The soil samples were collected for oil and grease
(O&G) analysis over time, the data for calculating
CO2 release were normalized to 1 kg of soil.
O&G analysis was performed to determine the
percentage of contaminant degradation and retention
by microorganisms in the soil samples at the initial
time point and at 30, 45, 60, and 90 days using the
ultrasound extraction method of USEPA 3550 C
(2007). This method is used for extracting nonvolatile
and semivolatile organic compounds from solids, such
as soils, sludge, and wastes. The ultrasonic process
ensures intimate contact of the sample matrix with the
extraction solvent. The soil was mixed with anhydrous
sodium sulfate (Na2SO4) and hexane solvent (C6H14)
for biodiesel extraction. A cell disruptor device (model
DES500, Unique, Brazil) with a macro tip at 99%
power was used for the extraction for 2 min. The
residual contents, calculated on a dry basis, were
obtained using the following equation:
M2  M1
O&Gð%Þ ¼  100 ð3Þ
M0
where O&G: oil and grease (%), M0: dry weight of the
soil sample used in the analysis (g), M1: weight of the
beaker (g), and M2: weight of the beaker with
contaminant extracted from the contaminated soil (g).
The result was obtained on a dry basis. To assess the
removal, the following equation was used without
considering the loss of volatile compounds:
O&Ginitial  O&Gfinal
Removalð%Þ ¼  100 ð4Þ
O&Ginitial
where O&Ginitial: initial concentration of oils and
greases and O&Gfinal: final concentration of oils and
greases.
To calculate the ratio of CO2 released and biode-
graded biodiesel, the ratio between the effective
release of CO2 (mg CO2) by effective biodiesel
bioremediation (g biodiesel) was considered. The
values used in this ratio were the final experiment time
(90 days) (Decesaro et al. 2016).
The effective release of CO2 was assessed by
subtracting the value of the control treatment (per-
formed according to item 2.3). While the effective
biodegradation was determined by excluding the
percentage of retention of the contaminant (biodiesel)
from the soil matrix. This percentage of retention was
determined from the experiment using sterile soil, thus
eliminating the hypothesis of biodiesel degradation
via microbial metabolism.
Data were tabulated, graphed, and analyzed by
analysis of variance (ANOVA), with a confidence
level of 95% (p \ 0.05). Statistica Software 8.0
(Statsoft 2007) was used for comparing the treatments.
Results and discussion
Biosurfactant production
The biosurfactant produced by B. methylotrophicus
reduced the surface tension of the whey-containing
culture medium from 43 to 30.2 mN/m, considering
the initial and final time points of fermentation. Three
production batches were required to obtain the neces-
sary amount of biosurfactants (3.84 g) for the biore-
mediation assay.
According to Decesaro et al. (2020), the biosurfac-
tant produced by B. methylotrophicus under the study
conditions is a lipopeptide named surfactin. According
123

to Jemil et al. (2017), surfactin is a potent surfactant
formed by a complex consisting of three peptide
synthetases (srfA-A, srfA-B, and srfA-C). The lower
surface tension values indicate that the biosurfactant
was produced. Surface tension helps to increase the
bioavailability of hydrophobic compounds, as it
allows interaction between phases due to the reduction
of repulsive interfacial forces (Varjani and Upasani
2016). Thus enabling the use of the biosurfactant
produced in the processes of bioremediation. The
biosurfactant has the property of reducing repulsive
forces, there may be a better emulsification between
contaminant and soil matrix, increasing the contact
surface areas and thus enabling better bioremediation
rates (Machado et al. 2020).
Bioremediation assay
Figure 1 shows the release of CO2 accumulated during
the bioremediation assay. In the first 12 days of the
assay, the respiratory activity in all the samples was
low (lower than 319 mg CCO2/kg). This effect could
be associated with the phase of adaptation of the
microorganisms to the contaminant and its concentra-
tion in the soil. After this period, higher CO2 release
was observed, inferring in the increases in the
microbial population and/or adaptation due to the
use of the present nutrient sources. Notably, the
highest increase in CO2 release occurred during the
Fig. 1 Cumulative CO2 release during 90 days of the bioremediation assay in biodiesel-contaminated soil. Note: Equal letters in the
same column indicate no significant difference at the 95% confidence level
123
Biodegradation
first 47 days of the assay. Considering the assay with
addition of 0.5% of biosurfactant, the release in this
period was 4155.91 mg CCO2/kg soil, while in the
following period (43 days) the release was only
2318.50 mg CCO2/kg soil, the other assays, except
the control assay, observed the same tendency. This
fact was also verified by Kreling et al. (2020) and
Kreling et al. (2018) in bioremediation of oily
residues, which can be attributed to the increased
microbial population in the medium and, conse-
quently, raising the CO2 release rates.
A higher cumulative value of CCO2 was noted in the
treatments with the addition of biosurfactants than in
the other treatments, showing a statistically significant
difference at the final time (p \ 0.05). This difference
was associated with the emulsifying action of the
biosurfactants added to the soil, which promoted
greater bioavailability of the contaminant (carbon
source), resulting in greater microbial growth and
greater release of CO2. If there is a comparison
between the assays, all experiments regardless of the
concentration of added biosurfactant (0.5%; 1.0%;
1.5%) showed higher values of CO2 release, when
compared to the natural attenuation and control
experiment.
The addition of 0.5% biosurfactant resulted in an
accumulated value of 6474.41 mg CCO2/kg of soil,
which is higher than the value obtained with the
addition of 1.0% biosurfactant (6399.02 mg CCO2/kg
Biodegradation
of soil) and that obtained with the addition of 0.1%
biosurfactant (5962.17 mg CCO2/kg of soil). However,
the Tukey test did not show significant differences
between the addition of the biosurfactant at concen-
trations of 1.0% and 0.5% (p [ 0.05), which is a good
result because it indicates that lower concentrations of
biocompounds values are associated with higher CO2
release, indicative of high microbial growth. Lesser
CO2 release was obtained with natural attenuation
than with the addition of 0.1% biosurfactant, reaching
a cumulative value of 5307.63 mg CCO2/kg of soil.
Low CO2 release (664.51 mg CCO2/kg soil) was
observed in the control assay. This result was expected
due to the low carbon content in the soil as well as the
nonexistence of the contaminant required for micro-
bial metabolism. The control treatment was performed
to assess the release of CO2 in the soil under normal
conditions.
The evaluation of CO2 release is an indirect way of
estimating the decarboxylation of organic compounds
that are degraded in the system (Margesin and
Schinner 2001). It is a proposed method for evaluating
the mineralization of a contaminant (biodiesel in this
study). Some studies, such as those of Silva et al.
(2009) and Meneghetti et al. (2012), have demon-
strated a relationship between CO2 release and organic
pollutant degradation.
Decesaro et al. (2016) evaluated the bioremediation
capacity of soil contaminated with oily compounds
using the biostimulation technique. The CO2 release in
biodiesel-contaminated soil biostimulated with phy-
cocyanin, extracted from the microalgae Spirulina
platensis, was 2997.3 mg of CCO2/300 g of soil, while
that in diesel-contaminated soil biostimulated with the
biomass of S. platensis was 2454.9 mg of CCO2/300 g
of soil; these findings differed from those of the
present study, in which there were lesser releases. This
difference occurred because the biosurfactant was
added in lower concentrations than that necessary to
attain the optimal conditions indicated for biostimu-
lation processes (C:N:P:K, 100:10:1:1) (Pope and
Matthews 1993; Riser-Roberts 1998; Beskoski et al.
2011).
According to Gupta (2020), the biodegradation of
biodiesel is faster than that of fuels of petrochemical
origin. This is because biodiesel is a mixture of
monoalkyl esters with high dissolution and it serves as
a sufficient carbon source for microorganisms (around
76.2% carbon, 12.6% hydrogen, and 11.2% oxygen by
weight).
The probable pathway of microbial biodegradation
consists of the lysis of methyl or ethyl ester by
esterases or lipases (first stage), producing a fatty acid
and an associated alcohol (second stage), and the
subsequent lysis of the fatty acid by the Krebs cycle
(third stage) (Vieira et al. 2006). In the second stage,
fatty acids are oxidized via b-oxidation and degraded
to acetic acid and a fatty acid consisting of two less
carbon atoms (Zhang et al. 1998). In this reaction, the
fatty acid is first converted to ester-coenzyme A (ester-
CoA). The produced ester-CoA is then oxidized, and
two carbon atoms at the end of the molecule are
divided to produce acetyl-CoA. This process of
shortening of the molecule continues until the initial
acid is completely degraded to acetyl-CoA (Chapelle
2000).
Figure 2 shows the removal and retention of
contaminants according to the concentrations of
biosurfactant added to the treatments with or without
sterilization, with the aim of evaluating the effect of
biosurfactant addition on contaminant retention in soil
over 90 days of bioremediation.
As shown in Fig. 2, in the treatments with nonster-
ile soil, the highest contaminant removal occurred in
the first 30 days; this was due to the larger phase of
microbial growth and, consequently, greater removal.
At the final time point (90 days), the removal of the
contaminant with the addition of 0.5% biosurfactant
was 57.25%; this was not significantly different from
the value obtained with the addition of 1.0% biosur-
factant (56.75%). The removal was 53.23% with the
addition of 0.1% biosurfactant; these values were
different from that obtained with natural attenuation
(41.17%). Contaminant removal was greater in the
treatments with the addition of the biosurfactant than
in the treatment with natural attenuation, with an
increase of 12.06%, 16.08%, and 15.58% being noted
with the addition of 0.1%, 0.5%, and 1.0% biosurfac-
tant, respectively. Thus, the biocompound affected
biodiesel degradation rates, even at concentrations
below the ideal C:N:P:K ratio. According to Cheng
and Mulla (1999), the recommended C:N:P ratio is
100:10:1 for soil bioremediation, indicative of its
performance as a biosurfactant. Beyond to the addition
of the biosurfactant, the potential of the soil auto-
chthonous microorganisms was evidenced, which are
123

Fig. 2 Biodegradation and cumulative retention of biodiesel in soil during the bioremediation assay: (a) total removal; (b) data analysis
at final time point (90 days). Note: Equal letters in the same column indicate no significant difference at the 95% confidence level
more effective when in microbial pools (Samaei et al.
2013, 2020).
Biosurfactants improve the bioremediation of oily
and poorly soluble compounds, such as biodiesel, by
two main mechanisms: the first includes the interac-
tion with poorly soluble contaminants and the
improvement of their transfer to the aqueous phase,
leading to increased mobility and bioavailability of the
contaminant (Burghoff 2012). While the other
involves its role in increasing the efficiency of
biodegradation. In fact, biosurfactants reduce surface
tension and thus increase the removal of oily com-
pounds, increasing their solubility and making them
more available for degradation (Hazra et al. 2012;
Nievas et al. 2008). In addition, biosurfactants also
influence the hydrophobicity of the bacterial cell
123
Biodegradation
surface, allowing hydrophobic substrates to associate
more easily with bacterial cells (Ayed et al. 2015).
In treatments with sterile soil, biodegradation is not
possible; therefore, the identified reduction in the
contaminant concentration may be due to retention in
the soil matrix, without indicating the occurrence of
degradation. Notably, at the initial time point, the
biodiesel retention values were between 6.0 and 8.0%,
varying according to the concentration of the added
biosurfactant. The higher the concentration of the
biosurfactant, the lower was the initial retention
(Fig. 2).
After 90 days, the contaminant removal was around
20% in all the treatments, with no significant differ-
ence among treatments, indicating that the biosurfac-
tant concentration did not affect the contaminant
retention in the soil; contaminant retention already
Biodegradation
occurred in the absence of the biosurfactant. This is an
important finding, revealing the importance of the
biosurfactant only in the solubilization of compounds
for the occurrence of biological processes, without
having any effect on physical processes.
Thomé et al. (2017) found that the fastest sorption
of soil contaminants occurred from 0 to 15 days and
the rate reduced in the final period of the assay; a
similar finding was observed in our study, where it was
found that up to the period of 15 days for experiments
with the addition of biosurfactant, the retention was
around 18%, reaching close to 20% at the end of the
trial. The same trend was observed for the assay with
natural attenuation, where from 0 to 15 days the
retention was 16.62% and after 90 days it reached the
maximum value of 20.75%. This phenomenon is
associated with the fact that the fastest interactions
between the mineral surface and the existing com-
pound in the medium occur in surface reactions.
Subsequent sorption processes are associated with the
migration of the contaminant to microscopic protru-
sions existing in the mineral particle, resulting in
further reduction in the contaminant at a later time
point. According to Weissenfels et al. (1992), there are
two types of retention kinetics of organic contami-
nants in soil: fast (surface) and slow (by migration to
deeper areas of the particle).
Therefore, an increase in the time of soil/contam-
inant contact results in a longer duration for the
migration of these compounds to less-accessible soil
areas, thereby making them a nondegradable fraction
in the contaminated soil (Reid et al. 2000). Conse-
quently, at the initial time point in our assay, there was
retention of the contaminant (about 18% in 15 days),
Table 1 Ratio of released CO2 and biodegraded biodiesel (mg CO2/g of biodiesel) at the final time point in the bioremediation assay
Treatment Effective CO2 release (mg of Effective biodiesel biodegradation
C–CO2/kg of soil) (g of biodiesel/kg of soil)
Biosurfactant 5297.67 ± 44.43 66.60 ± 1.68
0.1%
Biosurfactant 5809.90 ± 39.67 72.92 ± 0.63
0.5%
Biosurfactant 5734.52 ± 41.28 71.67 ± 0.66
1.0%
Natural 4643.12 ± 44.09 40.84 ± 0.53
attenuation
a
Equal letters in the same column indicate no significant difference at the 95% confidence level
and the maximum value of around 20% was reached in
all the treatments with sterile soil.
The values indicative of the relationship between
effective CO2 release and effective biodiesel
biodegradation are provided in Table 1.
In the treatments with the addition of the biosur-
factant, the amount of CO2 released per gram of
degraded contaminant was close to 80 mg of CCO2/g
of biodiesel; this indicates that about 11% of the
carbon in biodiesel was transformed to CO2. In the
natural attenuation treatment, a higher value
(113.69 mg of CCO2/g of biodiesel) was observed,
indicating about 8% transformation. This led to the
hypothesis that the carbon from biodiesel is used for
microbial growth. Subsequently, the biomass of
microorganisms will also be mineralized and con-
verted to CO2 and water; however, this will occur at
another time point. Other processes, such as fermen-
tation and anaerobic respiration, may also be involved
due to the large amount of contaminants added,
hampering soil aeration.
Decesaro et al. (2016) found that in some treat-
ments, the conversion rate was above 200 mg CCO2/g
of biodiesel, but the concentration of the contaminant
was lower (40 g/kg) under conditions of greater
aerobiosis. Thus, it is possible to explain differentiated
biodegradation in processes in which a large amount
of biodiesel is present (Table 2).
In general, in this study, the biosurfactant acted in
two main ways in the soil: (1) increasing the
availability of the contaminant to microorganisms
and (2) increasing the surface hydrophobicity, thereby
allowing the hydrophobic contaminant to interact
more easily with bacterial cells (Machado et al. 2020;
Ratio of released CO2 and biodegraded
biodiesel (mg CO2/g of biodiesel)a
79.58 ± 1.89a
79.67 ± 0.78a
80.02 ± 0.84a
113.30 ± 1.70b
123

Table 2 Ratio of released CO2 and biodegraded biodiesel (mg CO2/g of biodiesel) at the final time point in the bioremediation assay
Treatment Effective CO2 release (mg of Effective biodiesel biodegradation
C–CO2/kg of soil) (g of biodiesel/kg of soil)
Biosurfactant: 5297.67 ± 44.43 66.60 ± 1.68
0.1%
Biosurfactant: 5809.90 ± 39.67 72.92 ± 0.63
0.5%
Biosurfactant: 5734.52 ± 41.28 71.67 ± 0.66
1.0%
Natural 4643.12 ± 44.09 40.84 ± 0.53
attenuation
a
Equal letters in the same column indicate no significant difference at the 95% confidence level
Patowary et al. 2018). Calvo et al. (2009) and Pacwa-
Plociniczak et al. (2011) also verified that this leads to
greater soil bioremediation. This effect is because the
limited bioavailability of oily compounds in a terres-
trial environment due to low water solubility or
interaction with the soil matrix often inibits hydrocar-
bon degradation in the soil (Harms and Bosma 1997).
The addition of nutrients (N, P, and K) for
accelerating the biodegradation processes of toxic
organic compounds reduces the interaction between
the soil and contaminant (Thomé et al. 2017).
However, during in situ biostimulation, there is greater
leaching of the contaminant, which varies according to
the soil moisture content. This expands the contam-
ination plume and creates a false impression of a
decrease in organic compounds in the soil, facilitating
the harmful effects of contamination. Therefore, the
in situ addition of biosurfactants for the bioremedia-
tion of areas contaminated with oily compounds, such
as biodiesel, is a promising technique and should be
studied further.
Conclusion
The bacterial lipopeptide biosurfactant produced via
submerged fermentation was efficient and showed
potential for use in the bioremediation of soils
contaminated with biodiesel. The bioremediation
treatment with insertion of the biocompound at a
concentration of 0.5% (w/w) provided an increase in
the biodegradation of the contaminant and in the
release of CO2, 57.25% and 6474.41 mg CCO2/kg of
soil, respectively, when compared to the experiment of
123
Biodegradation
Ratio of released CO2 and biodegraded
biodiesel (mg CO2/g of biodiesel)a
79.58 ± 1.89a
79.67 ± 0.78a
80.02 ± 0.84a
113.30 ± 1.70b
natural attenuation (41.17% and 5307.63 mg CCO2/kg
of soil). The addition of 1.0% of biosurfactant had no
significant effect during bioremediation when com-
pared to the experiment with the addition of 0.5% of
biocompound, over the 90 days. These facts are
important to be considered in the application of
in situ bioremediation technologies, showing that
these biocompound produced with raw materials from
agroindustrial residues can be used in these processes.
Author contributions AD and TSM: Conducting a research
and investigation process, specifically performing the
experiments, or data/evidence collection; Preparation, creation
and/or presentation of the published work, specifically writing
the initial draft (including substantive translation); Writing—
Review & Editing. AR and ÂCC: Conducting a research and
investigation process, specifically performing the experiments,
or data/evidence collection; Preparation, creation and/or
presentation of the published work, specifically writing the
initial draft (including substantive translation). BSM and IC:
Writing—Review & Editing. AT and LMC: Supervision and
Acquisition of the financial support for the project leading to this
publication. Writing—Review & Editing.
Funding The authors are pleased to acknowledge the
Coordination of Improvement of Higher Education Personnel
(CAPES—Finance Code 001), Foundation of Support to the
Research of the State of Rio Grande do Sul (FAPERGS) and
University of Passo Fundo (UPF) for the financial support.
Declarations
Conflict of interest The authors declare that they have no
conflict of interest.
Biodegradation
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