Aquatic toxicity and biodegradability of a surfactant produced by Bacillus subtilis ICA56

Prévia do material em texto

Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=lesa20
Journal of Environmental Science and Health, Part A
Toxic/Hazardous Substances and Environmental Engineering
ISSN: 1093-4529 (Print) 1532-4117 (Online) Journal homepage: https://www.tandfonline.com/loi/lesa20
Aquatic toxicity and biodegradability of a
surfactant produced by Bacillus subtilis ICA56
Darlane W. F. De Oliveira, Alejandro B. Cara, Manuela Lechuga-Villena,
Miguel García-Román, Vania M. M. Melo, Luciana R. B. Gonçalves & Deisi A.
Vaz
To cite this article: Darlane W. F. De Oliveira, Alejandro B. Cara, Manuela Lechuga-Villena,
Miguel García-Román, Vania M. M. Melo, Luciana R. B. Gonçalves & Deisi A. Vaz (2017) Aquatic
toxicity and biodegradability of a surfactant produced by Bacillus�subtilis ICA56, Journal of
Environmental Science and Health, Part A, 52:2, 174-181, DOI: 10.1080/10934529.2016.1240491
To link to this article: https://doi.org/10.1080/10934529.2016.1240491
Published online: 28 Oct 2016.
Submit your article to this journal 
Article views: 218
View related articles 
View Crossmark data
Citing articles: 13 View citing articles 
https://www.tandfonline.com/action/journalInformation?journalCode=lesa20
https://www.tandfonline.com/loi/lesa20
https://www.tandfonline.com/action/showCitFormats?doi=10.1080/10934529.2016.1240491
https://doi.org/10.1080/10934529.2016.1240491
https://www.tandfonline.com/action/authorSubmission?journalCode=lesa20&show=instructions
https://www.tandfonline.com/action/authorSubmission?journalCode=lesa20&show=instructions
https://www.tandfonline.com/doi/mlt/10.1080/10934529.2016.1240491
https://www.tandfonline.com/doi/mlt/10.1080/10934529.2016.1240491
http://crossmark.crossref.org/dialog/?doi=10.1080/10934529.2016.1240491&domain=pdf&date_stamp=2016-10-28
http://crossmark.crossref.org/dialog/?doi=10.1080/10934529.2016.1240491&domain=pdf&date_stamp=2016-10-28
https://www.tandfonline.com/doi/citedby/10.1080/10934529.2016.1240491#tabModule
https://www.tandfonline.com/doi/citedby/10.1080/10934529.2016.1240491#tabModule
Aquatic toxicity and biodegradability of a surfactant produced by Bacillus subtilis
ICA56
Darlane W. F. De Oliveiraa, Alejandro B. Carab, Manuela Lechuga-Villenab, Miguel Garc�ıa-Rom�anb, Vania M. M. Meloc,
Luciana R. B. Gonçalvesa, and Deisi A. Vaz b
aDepartamento de Engenharia Qu�ımica, Universidade Federal do Cear�a, Fortaleza, CE, Brazil; bDepartment of Chemical Engineering, Faculty of Sciences,
University of Granada, Granada, Spain; cDepartamento de Biologia, LemBiotech, Laborat�orio de Ecologia Microbiana e Biotecnologia, Universidade
Federal do Cear�a, Fortaleza, CE, Brazil
ARTICLE HISTORY
Received 15 June 2016
Accepted 13 September 2016
ABSTRACT
In this work, the environmental compatibility of a biosurfactant produced by a Bacillus subtilis strain
isolated from the soil of a Brazilian mangrove was investigated. The biosurfactant, identified as surfactin, is
able to reduce surface tension (ST) to 31.5 § 0.1 mN m¡1 and exhibits a lowcritical micelle concentration
(CMC) value (0.015 § 0.003 g L¡1). The highest crude biosurfactant concentration (224.3 § 1.9 mg L¡1)
was reached at 72 h of fermentation. Acute toxicity tests, carried out with Daphnia magna, Vibrio fischeri
and Selenastrum capricornutum indicated that the toxicity of the biosurfactant is lower than that of its
chemically derived counterparts. The results of the biodegradability tests demonstrated that the crude
surfactin extract was degraded by both Pseudomonas putida and a mixed population from a sewage-
treatment plant, in both cases the biodegradation efficiency being dependent on the initial concentration
of the biosurfactant. Finally, as the biodegradation percentages obtained fall within the acceptance limits
established by the Organization for Economic Co-operation and Development (Guidelines for Testing of
Chemicals, OECD 301E), crude surfactin can be classified as a “readily” biodegradable compound.
KEYWORDS
Biosurfactant; environmental
compatibility; OECD
guidelines; saponin; surfactin
Introduction
Biosurfactants are surface-active compounds mainly produced
by bacteria, fungi and yeasts, although they can also be
extracted from plants and animals.[1,2] As amphiphilic com-
pounds, they exhibit pronounced surface and emulsifying activ-
ities and comprise a wide range of chemical structures with
diverse interfacial properties and physiological functions.[3]
In recent years, there has been an increasing interest in
microbial biosurfactants for several reasons. First, due to their
natural origin, biosurfactants are usually reported as relatively
nontoxic and readily biodegradable substances.[4] Second, bio-
surfactants have unique structures that are just starting to be
appreciated for their wide range of potential applications, from
biotechnology to environmental clean-up.[5] Finally, their sta-
bility under extreme conditions of pH, temperature and salin-
ity, allows their application in several industrial processes.
Although biosurfactants are generally considered “environmen-
tally friendly”, their ecological risk assessment is seldom stud-
ied.[6] To our knowledge, only a few studies on ecotoxicity of
biosurfactants have been carried out until now,[7–9] rhamnoli-
pids being the most extensively studied.[10]
It is well known that the effect of surface active agents on
the environment is dependent on several factors, their concen-
tration being the most important one. Even at very low
concentrations, surfactants seem to bind to membranes, affect-
ing some of their properties, such as permeability. At higher
concentrations, more drastic effects have been reported, such
as membrane lysis and fusion.[11] Van Hamme et al.[12] found
that at concentrations higher than their critical micelle
concentration (CMC), the toxic effect of biosurfactants on
microorganisms is a possible cause of bioremediation inhibi-
tion. However, despite exhibiting selective toxicity towards
specific pure cultures, biosurfactants may have a limited inhib-
itory impact on remediation systems involving diverse indige-
nous microbial populations.[4]
Not only toxicity but also biodegradation is what determines
the persistence and ultimate fate of biosurfactants in aquatic
and terrestrial ecosystems.[13] Hence, both the biodegradation
rate and the degree of toxicity are the data needed to establish
the potential impact of one specific compound on the biota of a
given ecosystem, and to regulate the uses of these substan-
ces.[14,15] The most preeminent group of of internationally
accepted biodegradation screening tests are the Guidelines for
the Testing of Chemicals issued by the Organization for Eco-
nomic Co-operation and Development (OECD), which play an
important role in the EU environmental classification of chemi-
cals and in the assessment of their environmental risk.[16]
The aim of this work is to study the environmental compati-
bility of a biosurfactant produced in our laboratory by Bacillus
subtilis ICA56. For that purpose, aquatic toxicity and
biodegradability tests were conducted using standardized tests,
and the experimental results were compared with those found
CONTACT Deisi A. Vaz deisiav@ugr.es Department of Chemical Engineering, Faculty of Sciences, University of Granada, Avda. Fuentenueva s/n, 18071 Granada,
Spain.
© 2017 Taylor & Francis Group, LLC
JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART A
2017, VOL. 52, NO. 2, 174–181
http://dx.doi.org/10.1080/10934529.2016.1240491
http://dx.doi.org/10.1080/10934529.2016.1240491
in literature for other chemical and microbial surfactants. The
toxicity of saponin from Quillaja Bark, which is considered a
generally recognized as safe (GRAS) substance by the by the US
Food and Drug Administration, was also evaluated.
Material and methods
Microorganism
B. subtilis ICA56, isolated from the soil of a Brazilian mangrove
(Icapui, Cear�a, Brazil), was kindly provided by the “Laborat�orio
deEcologia Microbiana e Biotecnologia” of the Federal Univer-
sity of Cear�a (Brazil). The 16S rRNA gene sequence of ICA56 is
available in the GenBank (accession number KM235112). The
strain was kept in a culture medium composed of 15.0 g L¡1
agar, 5.0 g L¡1 peptone, 5.0 g L¡1 glucose and 2.5 g L¡1 yeast
extract (APGE) at 4�C for a maximum of 2 weeks.[17]
Culture medium
The culture medium used for biosurfactant production was
adapted from Mor�an et al.[18] and was composed of 10.0 g L¡1
of glucose, 5.0 g L¡1 of yeast extract, 1.0 g L¡1 of (NH4)2SO4,
6.0 g L¡1 of Na2HPO4, 3.0 g L¡1 of KH2PO4, 2.7 g L¡1 of NaCl
and 0.6 g L¡1 of MgSO4¢7H2O. The pH of the culture medium
was adjusted to 7.0 with 3.0 M NaOH or 3.0 M HCl. After
autoclaving at 110�C for 10 min, the culture medium was
supplemented with 0.1% (v/v) of a sterile solution of trace
elements, as described by Freitas de Oliveira.[19]
Inoculum
B. subtilis ICA56 was firstly inoculated into APGE plates and
incubated at 30�C for 24 h. After this period, three loops were
transferred to 250-mL Erlenmeyer flasks containing 50 mL of
culture medium and 0.1% of trace element solution.[19] The
flasks were then incubated at 30�C and 180 rpm for 24 h, and
finally, its optical density was adjusted to 0.2 at 600 nm with
the same culture medium but without glucose.[18]
Biosurfactant production
The biosurfactant used in this work was produced by B. subtilis
ICA56 in a submerged fermentation system (SF). Erlenmeyer
flasks containing the culture medium described previously and
a 10% of inoculum were incubated in a rotary shaker at 30�C
and 150 rpm, as described by França et al.[17] After 72 h of fer-
mentation, bacterial cells were harvested by centrifugation at
9,000 rpm for 20 min at 4�C, and its concentration was deter-
mined as described by Freitas de Oliveira et al.[19] Once the sur-
face tension (ST) of the cell-free supernatant was measured, its
pH was adjusted to 2.0 by addition of 6 M HCl, and it was left
to stand overnight at 4�C. The biosurfactant precipitated was
separated from the liquid by centrifugation (9,000 rpm for
20 min at 4�C), and lyophilized. The so called “crude biosurfac-
tant” was weighed and stored at ¡18�C for further use. To fur-
ther purify the crude biosurfactant it was dialyzed overnight
against demineralized water in a Spectra/Por� Dialysis Mem-
brane (cut-off 6,000–8,000 Da, Spectrum Laboratories Inc.,
Houston, TX, USA). This purified biosurfactant was precipi-
tated again from the dialyzed solution by HCl addition, lyophi-
lized, and used for structural characterization (FTIR and mass
spectra). To follow the kinetics of the fermentation process,
biosurfactant and glucose concentrations, as well as ST, were
determined throughout the 72 h of the experiment.
Glucose concentration
The concentration of glucose on the cell-free supernatant was
measured by HPLC using a Waters chromatograph (Waters,
Milford, MA, USA), equipped with a refractive index detector
(Model 2414, Waters), a Supelcogel C610H (30 £ 7.8 mm) col-
umn and a Sigma-Aldrich (St. Louis, MO, USA) pre-column (5
£ 4.6 mm). Ultrapure water (Simplicity 185, Millipore©) with
0.1% v/v of H3PO4 was used as elution solvent with a flow rate
of 0.5 mL min¡1. The carbohydrates present in the samples
were identified by comparing the retention times with those
found for carbohydrate standards, as described by Freitas de
Oliveira et al.[19]
Emulsification index
The emulsifying activity of the biosurfactant was evaluated as
described by Cooper and Goldenberg.[20] To do this, 2 mL of
engine oil (REPSOL, Madrid, Spain) and the same volume of
cell-free supernatant or crude biosurfactant solution were
vortexed at high speed for 2 min in a test tube and kept at 30�C
for 24 h. The emulsification index (E24) was calculated as the
height of the emulsified layer (mm) divided by the total height
of the liquid column (mm) at the end of this period. All tests
were carried out in triplicate.
Interfacial properties
ST of crude biosurfactant solutions was measured at 25�C by
the Wilhelmy Plate method in a KRUSS K11 tensiometer
(KR€USS GmbH, Hamburg, Germany). CMC was determined
from ST data by finding the break point in the surface tension
vs. log of concentration curve. Interfacial tension (IT) between
crude biosurfactant solutions and engine oil (REPSOL) was
measured at 25�C in a pendant drop tensiometer (KSV CAM
200, KSV Instruments Ltd., Finland). All measurements were
done in triplicate.
Structural characterization
The chemical structure of the purified biosurfactant was
studied by Fourier-Transform Infrared Spectroscopy (FTIR)
and mass spectrometry analysis. The FTIR spectrum of the
biosurfactant was recorded in a JASCO FTIR-6200 spectrome-
ter (JASCO Analytical Instruments, Tokyo, Japan) with a
triglycine sulfate (TGS) detector. The samples were prepared
by dispersing the purified biosurfactant in a potassium bro-
mide matrix. To fully characterize the biosurfactant, the par-
tially purified extract was fractionated by chromatography,
and the different fractions were identified by mass spectrome-
try, following the procedure described by Moya-Ramirez
et al.[21] The corresponding measurements were conducted in
JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART A 175
an UPLC Waters Acquity H-Class chromatograph (Waters
Corporation, Milford, MA, USA), equipped with a Waters
UPLC BEH C-18 column and coupled to a mass spectrometer
(Waters Xevo-TQ-S). Data were acquired in the selected ion
recording mode, at the molecular weights detected in the analy-
sis of the surfactin standard (Sigma-Aldrich, �98% of purity).
Acute toxicity tests
Acute toxicity of the biosurfactant produced by B. subtilis
ICA56, as well as that of saponin from Quillaja Bark (Sigma-
Aldrich), was evaluated by three different techniques: (i) the
LUMIStox� 300 test, which employs the luminescent bacte-
rium Vibrio fischeri, (ii) a 24-h immobilization test with
Daphnia magna, and (iii) a 72-h algal growth inhibition test
with Selenastrum capricornutum.
In acute toxicity assays carried out with the marine bacteria
V. fischeri NRRL-B-11177, the reduction of the luminous inten-
sity emitted by the bacteria after being exposed to the test sub-
stance was measured. Bioluminescence was determined with
the LUMIStox� 300 device, according to the UNE-EN ISO
11348-2 guideline, at 15 and 30 min exposure times. The toxic-
ity of the tested substance was expressed in terms of its EC20
and EC50 values (mg L
¡1), which correspond to the surfactant
concentrations that cause a 20% and 50% reduction in the
intensity of luminescence emitted by the bacterial suspension,
respectively.
The 24-h immobilization test with D. magna was performed
with the Crustacean Toxicity Screening Test for Freshwater
Daphtoxkit FTM Magna (MicroBioTests Inc., Gent, Belgium),
which conforms to the standards of international organizations
(OECD, ISO, USEPA and ASTM). After 24 h of exposure, the
number of immobilized specimens was determined visually,
and EC50 was calculated as the amount of surfactant, which
inhibits the mobility of 50% of Daphnia population. No feeding
and no aeration were provided during the tests.
The 72-h growth inhibition test with the algae S. capricornu-
tum (renamed as Pseudokirchneriella subcapitata) was carried
out using the commercial test Algaltoxkit FTM (MicroBioTests
Inc.), which follows the OECD Guideline no. 201. Vials con-
taining the biosurfactant, together with the nutrient medium
and a suspension of the algae, were kept at 20 § 2�C under a
constant and uniform illumination. The toxic effect of the bio-
surfactant on microalgal growth was evaluated by measuring
the optical density of the culture at 670 nm at 24, 48 and 72 h
of exposure. EC50 values were calculated using linear regression
analysis based on the dose-response curves. At least two
replicates of each assay were carried out.
Ready biodegradability tests
Biodegradability tests were conducted using a pure culture of
Pseudomonas putida (UNE-EN ISO 10712:1996)and a mixed
bacterial population (OECD 301 E test for ready biodegradabil-
ity evaluation).
For the first one, the P. putida CECT 324 strain (Spanish
Type Culture Collection, Valencia, Spain) was used. A bacterial
stock was inoculated into a 250-mL Erlenmeyer flask contain-
ing 100 mL of biosurfactant solution, and the flasks were
incubated at 30�C and 100 rpm in darkness for 3 days. The deg-
radation of the biosurfactant by P. putida was followed by mea-
suring the dissolved organic carbon (DOC) with a TOC
analyzer Shimadzu TOC-V CHS (Shimadzu, Kyoto, Japan).
The biodegradation efficiency (Ef, %) was calculated according
to the following equation:
Ef D DOCi ¡DOCfDOCi £ 100 (1)
where DOCi and DOCf are the dissolved organic carbon
concentrations determined at the beginning and at the end of
the incubation period, respectively. The procedure used to pre-
pare the bacterial stock and mineral medium can be found
elsewhere.[22]
In the ready biodegradability test, the biosurfactant solution
is inoculated with the effluent of a secondary treatment of a
sewage-treatment plant, which operates with the active sludge
process, and incubated at 25�C for 28 days in darkness at
125 rpm. DOC concentration was also used to monitor the bio-
degradation process. A compound is regarded as “readily bio-
degradable” if more than 70% of it is degraded within 28 days.
At least two replicates were carried out for all the assays.
Results and discussion
Biosurfactant production and characterization
Biosurfactant production was monitored throughout the fer-
mentation by determining crude biosurfactant, glucose and cell
concentrations, as well as pH and ST of the cell-free superna-
tant (Fig. 1). As can be seen in Figure 1, ST of the culture media
was drastically reduced to 31.5 § 0.1 mN m¡1 within the first
10 h of the process, which coincides with the exponential phase
of growth of B. subtilis ICA56, and remained almost constant
after that. After 20 h, glucose was completely consumed. The
maximum biosurfactant concentration was 224.3 §
1.9 mg L¡1, attained at 72 h of fermentation. Therefore, the
experimental data suggest that under these conditions, the
biosurfactant production is associated with both exponential
and stationary growth phases of the microorganism. The pH of
the culture media varied from 7.2 § 0.1, at the beginning of the
fermentation, to 6.84 § 0.44 at 72 h.
Figure 1. Time course of (!) surface tension and (^) biosurfactant (mg L¡1), (�)
glucose (g L¡1), and (&) biomass concentration (g L¡1) throughout fermentation
by B. subtilis ICA56.
176 D. W. F. DE OLIVEIRA ET AL.
The production of biosurfactants from glucose by B. subtilis
is well documented in literature.[17,23,24] França et al.[17] used
the same microorganism as in this work to produce lipopepti-
des from agro-industrial wastes. For comparison purposes
only, they also carried out fermentations with 20 g L¡1 of glu-
cose (control assay). The concentration of biosurfactant in this
control experiment was measured at 48 h, being 410 §
160 mg L¡1, i.e., approximately twice as that obtained in this
work with 10 g L¡1 of glucose.
Interfacial properties of the biosurfactant from B. subtilis
ICA56
Table 1 shows the values of critical micelle concentration, sur-
face tension, interfacial tension and emulsification index deter-
mined at 25�C with solutions of the crude biosurfactant.
Although the purity and homologue distribution of surfactin
may differ from one study to another, our experimental results
are in good agreement with those reported by França et al.[17]
and Deleu et al.[25] It is worth mentioning that the value of the
minimal ST, determined at the CMC of the biosurfactant, is
lower than 35 mN m¡1, which is an important feature. Accord-
ing to Mulligan,[26] a good biosurfactant must be able to lower
the ST of water from 72 to 35 mN m¡1. Furthermore, consider-
ing that commercial surfactin presents CMC values ranging
from 0.0078 to 0.0207 g L¡1,[27] the value obtained in this work
(0.015 g L¡1) seems to be very satisfactory.
Chemical structure of the biosurfactant produced by ICA56
Purified biosurfactant was analyzed by Fourier-Transform
Infrared Spectroscopy and Mass Spectrometry. The analysis of
the biosurfactant FTIR spectra reveals a great similarity to that
of a surfactin standard (Sigma-Aldrich, �98% of purity)
(Fig. 2). The characteristic absorbance bands of peptides can be
clearly seen at 3,430 cm¡1 (NH-stretching mode), 1,655 cm¡1
(CO–N stretching mode), and 1,534 cm¡1 (N–H deformation
mode combined with C–N stretching mode). The presence of
aliphatic chains (–CH3; –CH2–), represented by the bands
between 3000–2800 cm¡1 and 1465–1368 cm¡1 can also be
observed. The band observed at 1730 cm¡1 is characteristic of
an ester carbonyl group. The existence of an aliphatic chain
together with a peptide moiety indicates that the biosurfactant
belongs to the lipopeptide group. Similar results have previ-
ously been reported by Pereira et al.[28] and Faria et al.[29] The
different homologues that can be found in the commercial sur-
factin standard were also detected in the purified biosurfactant,
and at the same retention times, according to the mass spec-
trometry results (data not shown). Hence, for the first time, the
biosurfactant produced by the B. subtilis strain ICA56 could be
identified as surfactin. This result was expected since B. subtilis
strains are well-known producers of surfactin.[30]
Toxicity of the biosurfactant produced by B. subtilis ICA 56
to aquatic organisms
Acute toxicity test with Vibrio fischeri
Toxicity is the degree to which a specific compound or a mix-
ture is capable of causing damage or death in living organisms.
The toxic effects of the crude biosurfactant from B. subtilis
ICA56 on the marine bacterium V. fischeri were evaluated by
determining the percentage of inhibition of bacterial light emis-
sion at different biosurfactant concentrations. The results are
shown in Table 2,[31-34] which presents the EC20 and EC50 val-
ues after 15 and 30 min of exposure. The toxicity of saponin, a
plant-derived biosurfactant, and some synthetic surfactants
(taken from literature) is also shown in Table 2. It can be seen
that independently of the exposure time, the EC20 and EC50 val-
ues of the crude biosurfactant and saponin are similar, and sig-
nificantly higher than those reported for synthetic surfactants.
Even alkyl polyglucosides, which are obtained from natural
sources and generally considered as non-toxic, exhibited a
higher toxicity on V. fischeri than surfactin from B. subtilis
ICA56. Pietra-Ottlik et al.,[34] studying the toxicity of gemini
surfactants, reported that the surfactants that were less toxic to
V. fischeri exhibited a EC50-15min value of 980 mg L
¡1, which
corresponds to approximately 1/50 of its CMC. Interestingly,
Lima et al.[31] found that the EC20 values of lipopeptides were
about 2 or 3 times its CMC, which is in good agreement with
the results reported in this work for surfactin.
It is well known that the toxic effects of the surface-
active agents on biological membranes are clearly dependent
on its concentration in the aqueous media. Deleu et al.[25]
and Francius et al.,[35] when investigating the effect of sur-
factin on artificial membrane integrity, found that, at low
concentrations, surfactin inserts exclusively in the outer
leaflet of the membrane inducing only a limited perturba-
tion. However, further addition of surfactin leads to a
Table 1. Interfacial properties of the crude biosurfactant produced by B. subtilis
ICA56 at 25�C.
CMC (g L¡1) ST (mN m¡1) IT (mN m¡1) E24 (%)
0.015 § 0.003 33.93 § 0.77a 10.63 § 0.08b 62.9§ 0.1c
aST was determined at CMC of the crude biosurfactant.
bIT was determined with the engine oil REPSOL 50501 TDI 5W40.
cE24 value corresponds to the measurement carried out with 1 g L¡1 of the biosur-
factant and the engine oil.
Figure 2. FTIR spectra of the partially purified biosurfactant produced by Bacillus
subtilis ICA 56 and of a commercialsurfactin standard.
JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART A 177
transient permeabilization of the membrane, or even the
complete disruption and solubilization of the lipid bilayer
with formation of mixed micelles. Similar effects of sapo-
nins have also been reported.[36–39]
Acute toxicity test with Daphnia magna
D. magna is accepted in several countries as a test organism to
monitor the efficacy of wastewater treatment systems, as well as
to establish the acceptable concentrations of pollutants.[40]
Table 3 [41-43] summarizes the values of EC50 of the crude sur-
factin produced by B. subtilis ICA56 and saponin, as well as
those of different chemical surfactants, obtained with this stan-
dard test. A lower EC50 value means a higher toxicity of the
tested compound. Again, the EC50 values of both the biosurfac-
tant from B. subtilis ICA56 and saponin, after 48 h of exposure,
are similar, the result for the former being slightly higher than
that for the latter. In any case, the EC50 of the biosurfactant
produced in this study is very high (11 times its CMC), which
indicates that no acute effects on D. magna are expected at
environmentally relevant concentrations. Once more, and com-
parable to what happened with the V. fischeri test, EC50 values
of chemical surfactants are lower than those found for the bio-
surfactants. For example, the values of EC50 found for crude
surfactin and saponin are more than 8 times higher than those
reported for sodium dodecyl sulfate (SDS), an anionic surfac-
tant commonly used in products for personal care.
Toxicity test with the unicellular alga Selenastrum
capricornutum
Due to its sensitivity to a great variety of hazardous sub-
stances, the green alga S. capricornutum is used as an indi-
cator of phytotoxicity of biosurfactants.[44] In this test, we
found that the biosurfactant concentration needed to pro-
duce a 50% growth inhibition (72 h EC50) was 49.3 and
36.5 mg L¡1, for the lipopeptide and saponin, respectively
(Fig. 3). Hence, EC50 values found for crude surfactin with
S. capricornutum are from 3 to 4 times lower than those
measured with D. magna. Therefore, our experimental data
suggest that the freshwater green alga is more sensitive to
crude surfactin than D. magna.
The EC50 values found for both biosurfactants, particu-
larly for surfactin, are also higher than those reported for
nonionic synthetic surfactants, which range from 2 to
50 mg L¡1,[45] which indicates a higher inhibitory effect of
nonionic synthetic surfactants on the growth of S. capricor-
nutum. Meanwhile, as regards the anionic surfactants, it is
remarkable that the EC50 value of linear alkylbenzene sulfo-
nate (LAS), the world’s largest-volume synthetic surfactant
in 2008,[46] ranged from 10 to 151 mg L¡1,[33,45] which is
relatively close to those found in this work for the crude
surfactin and saponin.
Table 2. Ecotoxicity of surface-active agents to the luminescent bacterium V. fischeri.
Surfactant EC20-15 min (mg L
¡1) EC50-15 min (mg L¡1) EC20-30 min (mg L¡1) EC50-30 min (mg L¡1) Reference
Surfactin from ICA 56 179.8 912.4 159.4 848.2 This work
Saponin from Quillaja Bark (Sigma) 176.3 652.7 218.3 578.4 This work
LBBMA 155 (lipopeptide) ND ND 352.5 ND [31]
LBBMA 201 (lipopeptide) 613.2 ND 523.0 ND [31]
Rhamnolipids from P. aeruginosa ND ND ND 45.0 [32]
Linear alkylbenzene sulfonate (LAS) 18.5 33.5 11.3 32.0 [22]
Sodium dodecyl sulfate (SDS) 29.7 ND 25.27 ND [31]
Alkyl polyglucosides (APG) 3.9 to 10.0 14.0 to 29.0 4.0 to 7.3 14.0 to 25.0 [33]
Dicephalic ammonium surfactants ND 2.6 to 980.0 ND ND [34]
ND: non determined.
Table 3. Ecotoxicity of surface-active agents to the microcrustacean Daphnia
magna.
Surfactant EC50 (mg L
¡1) Ratio EC50/CMC Reference
Surfactin from ICA56 170.1 11.3 This work
Saponin from Quillaja
Bark (Sigma)
128.4 0.3 This work
LAS 0.7–53 ND [41]
Nonylphenol ethoxylates 2.5–19.1 ND [42]
APG 29–111 0.1–0.3 [33]
SDS 14.5–16.2 ND [40]
Lysine based surfactants
(cationic surfactant)
1.5–5.5 mM 0.006–0.005 [43] Figure 3. Dose-response curves of the assays carried out with Selenastrum capri-
cornutum: (a) experiments with crude surfactin produced by B. subtilis ICA56 and
(b) saponin.
178 D. W. F. DE OLIVEIRA ET AL.
Biodegradability assays
Biodegradation by Pseudomonas putida
The biodegradability of the biosurfactant from B. subtilis ICA56
was evaluated in a 72-h test with P. putida, an almost ubiqui-
tous bacterium in limnic surface waters and soils.[47] Figure 4
shows the biodegradability-concentration profile of crude
surfactin obtained at 72 h of test. The experimental data put in
evidence that biosurfactant biodegradation depends on the
initial biosurfactant concentration, the highest biodegradability
values (>65%) being found at concentrations lower than
100 mg L¡1. A drastic decrease on the biodegradability (from
65 to 10%) was observed for biosurfactant concentrations in
the range from 100 to 200 mg L¡1.
Lima et al.[31] studied the biodegradability of microbial and
synthetic surfactants by a respirometric technique. According
to these authors, although both lipopeptides and SDS were bio-
degraded by Pseudomonas sp., the extent of SDS degradation
was significantly lower (about 10 times) in comparison to that
observed for the biosurfactants. Jurado et al.[48] reported bio-
degradation efficiencies of amine oxides by P. putida higher
than 50% at concentrations lower than 30 mg L¡1. Singh
et al.[49] found that even at concentrations below its CMC, the
growth of Pseudomonas aeruginosa was completely inhibited
by the presence of SDS. Hence, according to the published
data, surfactin from ICA56 seems to be more easily biodegrad-
able than its counterparts.
Biodegradation of surfactin by activated sludge
microorganisms
Although biodegradability is generally tested using pure bacte-
rial cultures, it is also useful to evaluate the degradation of a
chemical compound by a complex microbial community.
Figure 5 shows the evolution of DOC throughout the biodegra-
dation assays with activated sludge microorganisms at different
concentrations of crude surfactin. Independently of the initial
biosurfactant concentration, the experimental data show the
existence of an initial period of acclimatization of the microor-
ganisms (lag phase) in the first 7 h of the process, during which
the DOC values remained nearly constant. This behavior was
previously reported by Jurado et al.[50] in static biodegradability
tests carried out with conventional surfactants. After this
period, DOC was significantly reduced over the 28-day course
of the test. Interestingly, at 216 h (9 days) of experiment, biode-
gradability achieved values higher than 65%. In any case, there
always seems to be a residual DOC concentration that cannot
be reduced by the action of the microorganism. To estimate
this value, the experimental data were fitted to a pseudo-first-
order model (see Fig. 5), according to which this residual con-
centration was always lower than 3%.
As the biodegradation percentages obtained fall within the
acceptance limits established by the OECD (OECD Guidelines
301E), crude surfactin can be classified as a “readily” biode-
gradable compound. In a similar test, Hirata et al.[51] followed
the biodegradation process of surfactin by measuring the Bio-
chemical Oxygen Demand (OECD Guidelines 301C). Accord-
ing to these authors, a 61% biodegradation percentage was
reached for a surfactin solution of 25 mg L¡1.
Table 4 summarizes the characteristic parameters of the bio-
degradation process, particularly the average biodegradation
rate (Vm), the half-life (t1/2), and the initial (CODi) and residual
chemical oxygen demand (CODf). It should be noted that the
half-life (defined as the time needed to reduce the initial con-
centration of surfactant to a half) varied from 7 h (10 mg L¡1
of biosurfactant) to 3 days (50, 100 and 200 mg L¡1).
Conclusions
In this work, the environmental properties of crude surfactin
produced by B. subtilis ICA56 are reported. Acute toxicitytests,
carried out with V. fischeri, D. magna, and S. capricornutum,
show that the toxicity of the biosurfactant is lower than that
reported for conventional surfactants. The results of the tests
with P. putida indicate that the biodegradation process depends
Figure 4. Biodegradation of crude biosurfactant solutions by P. putida. The stan-
dard deviation of measurements is lower than 3.1%.
Figure 5. Time course of dissolved organic carbon as a function of incubation time
under conditions of the OECD screening test (OECD 301 E).
Table 4. Characteristic parameters of the biodegradation of the biosurfactant from
B. subtilis ICA56 by activated sludge microorganisms.
Biosurfactant concentration (mg L¡1)
Parameter 10 25 50 100 200
CODi (mg L
¡1) 5.74 11.30 20.68 43.16 88.32
CODf (mg L
¡1) 2.69 2.14 2.14 3.13 5.76
t1/2 (d) 7.00 7.00 3.00 3.17 2.92
Vm (%.d
¡1) 7.14 7.14 16.67 15.79 17.14
B (%) 53.15 81.04 89.65 92.75 93.48
CODi and CODf: initial and final chemical oxygen demand, respectively; t1/2: half-
life; Vm: average biodegradation rate; B: percentage reduction of COD.
JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART A 179
on the initial concentration of surfactin, the highest biodegrad-
ability values being found at concentrations lower than
100 mg L¡1. Finally, as the biodegradation percentage achieved
in this work exceeds the biodegradation level established by the
OECD Guidelines for Testing of Chemicals (OECD 301E), sur-
factin can be classified as a “readily” biodegradable compound.
Funding
The authors acknowledge the financial support of the Brazilian and Span-
ish Governments through project PHB2012-0277-PC, and of the Andalu-
sian Regional Government through project P10-TEP-6550. D.W.F.O. They
also acknowledge CNPq (Brazil) for the award of a scholarship and finan-
cial support.
ORCID
Deisi A. Vaz http://orcid.org/0000-0002-0866-9708
References
[1] Paria, S. Surfactant-enhanced remediation of organic contaminated
soil and water. Adv. Colloid Interf. Sci. 2008, 138(1), 24–58.
[2] Al-Wahaibi, Y.; Joshi S.; Al-Bahry, J.S.; Elshafie, A.; Al-Bemani, A.;
Shibulal, B. Biosurfactant production by Bacillus subtilis B30 and its
application in enhancing oil recovery. Colloids Surf. B 2014, 114,
324–333.
[3] Rodrigues, L.R.; Texeira, J.A. Biomedical and theurapeutic applica-
tions of biosurfactants. In Biosurfactants; Sen, R., Ed.; Landes Biosci-
ence and Springer ScienceCBusiness Media: New York, NY, 2010;
75–87.
[4] Silva, R.D.C.F.; Almeida, D.G.; Rufino, R.D.; Luna, J.M.; Santos, V.A.;
Sarubbo, L. A. Applications of biosurfactants in the petroleum indus-
try and the remediation of oil spills. Int. J. Mol. Sci. 2014, 15(7),
12523–12542.
[5] Sober�on-Chavez, G.; Maier, R.M. Biosurfactants: a general overview.
In Biosurfactants. From genes to applications; Sober�on-Chavez, G.,
Ed.; Springer: M€unster, 2011; 1–1.
[6] Galabova, D.; Sotirova, A.; Karpenkoy, E.; Karpenko, O. Role of
microbial Surface-active compounds in environmental protection. In
The Role of Colloidal Systems in Environmental Protection; Fanun,
M., Ed.; Springer: Berlin, 2014; 41–83.
[7] Franzetti, A.; Gandolfi, I.; Raimondi, C.; Bestetti, G.; Banat, I.M.;
Smyth, T.J.; Papacchini, M.; Cavallo, M.; Fracchia, L. Environmental
fate, toxicity, characteristics and potential applications of novel bioe-
mulsifiers produced by Variovorax paradoxus 7bCT5. Bioresour.
Technol. 2012, 108, 245–251.
[8] Edwards, K.R.; Lepo, J.E.; Lewis, M.A. Toxicity comparison of bio-
surfactants and synthetic surfactants used in oil spill remediation to
two estuarine species. Mar. Pollut. Bull. 2003, 46(10), 1309–1316.
[9] Ivshina, I.B.; Kuyukina, M.S.; Philp, J.C.; Christofi, N. Oil desorption
from mineral and organic materials using biosurfactant complexes
produced by Rhodococcus species. World J. Microbiol. Biotechnol.
1998, 14(5), 711–717.
[10] Mnif, I.; Ghribi, D. Microbial derived surface active compounds:
properties and screening concept. World J. Microbiol. Biotechnol.
2015, 31(7), 1001–1020.
[11] Helenius, A.; Simons, K. Solubilization of membranes by detergents.
Biochim. Biophys. Acta 1975, 415, 29–79.
[12] Van Hamme, J.D.; Ward, O.P. Influence of chemical surfactants on
the biodegradation of crude oil by a mixed bacterial culture. Can. J.
Microbiol. 1999, 45(2), 130–137.
[13] Raymond, J.W.; Rogers, T.N.; Shonnard, D.R.; Kline, A.A. A review
of structure-based biodegradation estimation methods. J. Hazard.
Mater. 2001, 84(2), 189–215.
[14] Sineva, A. Adsorption of synthetic surfactants from aqueous solu-
tions on natural adsorbents. In The Role of Colloidal Systems in Envi-
ronmental Protection; Fanun, M., Ed.; Elsevier: Amsterdam, 2014;
142–171.
[15] Hagner, M.; Penttinen, O.P.; Pasanen, T.; Tiilikkala, K.; Set€al€a, H.
Acute toxicity of birch tar oil on aquatic organisms. Agric. Food Sci.
2010, 19, 24–32.
[16] Guhl, W.; Steber, J. The value of biodegradation screening test results
for predicting the elimination of chemicals’ organic carbon in waste
water treatment plants. Chemosphere 2006, 63(1), 9–16.
[17] França, I.W.L.; Parente Lima, A.; Monteiro Lemos, J.A.; Faria
Lemos, C.G.; Melo, V.M.M.; Batista de Santana, H.; Gonçalves,
L.R.B. Production of a biosurfactant by Bacillus subtilis ICA56
aiming bioremediation of impacted soils. Catal. Today 2015, 255,
10–15.
[18] Mor�an, A.C.; Olivera, N.; Commendatore, M.; Esteves, J.L.; Si~neriz, F.
Enhancement of hydrocarbon waste biodegradation by addition of a
biosurfactant from Bacillus subtilis O9. Biodegradation 2000, 11(1),
65–71.
[19] Freitas de Oliveira, D.W.; França, I.W.L.; Felix, A.K.; Martins, J J.L.;
Giro, M.E. A.; Melo, V.M.M.; Gonçalves, L.R.B. Kinetic study of bio-
surfactant production by Bacillus subtilis LAMI005 grown in clarified
cashew apple juice. Colloids Surf. B. 2013, 101, 34–43.
[20] Cooper, D.G.; Goldenberg, B.G. Surface-active agents from two bacil-
lus species. Appl. Environ. Microbiol. 1987, 53(2), 224–229.
[21] Moya-Ram�ırez, I.; Altmajer Vaz, D.; Banat, I.M.; Marchant, R.;
Jurado Alameda, E.; Garc�ıa-Rom�an, M. Hydrolysis of olive mill waste
to enhance rhamnolipids and surfactin production. Bioresour. Tech-
nol. 2016, 205, 1–6.
[22] Lechuga Villena, M.; Fern�andez-Serrano, M.; Jurado, E.; Fern�andez-
Arteaga, A.; Burgos, A.; R�ıos, F. Influence of ozonation process on
the microbial degradation of surfactants. Procedia Engineering 2012,
42, 1038–1044
[23] Maass, D.; Moya Ram�ırez, I.; Garc�ıa Rom�an, M.; Jurado Alameda, E.;
Ulson de Souza, A.A.; Borges Valle, J.A.; Altmajer Vaz, D. Two-phase
olive mill waste (alpeorujo) as carbon source for biosurfactant pro-
duction, J. Chem. Technol. Biotechnol. 2016, 91(7), 1990–1997.
[24] Altmajer Vaz, D.; Gudi~na, E.J.; Jurado Alameda, E.; Teixeira, J.A.;
Rodrigues, L.R. Performance of a biosurfactant produced by a
Bacillus subtilis strain isolated from crude oil samples as com-
pared to commercial chemical surfactants. Colloids Surf. B.
2012, 89, 167–174.
[25] Deleu, M.; Nott, K.; Brasseur, R.; Jacques, P.; Thonart, P.; Dufr�ene, Y.
F. Imaging mixed lipid monolayers by dynamic atomic force micros-
copy. Biochim. Biophys. Acta 2001, 1513(1), 55–62.
[26] Mulligan, C.N. Environmental applications for biosurfactants. Envi-
ron. Pollut. 2005, 133(2), 183–198.
[27] Sigmaâ–Aldrich, Product details surfactin from Bacillus subtilis
(S3523), 2016. Available at http://www.sigmaaldrich.com/catalog/
product/sigma/s3523?langDes&regionDES (accessed Apr 2016).
[28] Pereira, J.F.; Gudi~na, E.J.; Costa, R.; Vitorino, R.; Teixeira, J.A.; Cou-
tinho, J.A.; Rodrigues, L.R. Optimization and characterization of bio-
surfactant production by Bacillus subtilis isolates towards microbial
enhanced oil recovery applications. Fuel 2013, 111, 259–268.
[29] Faria, A.F.; Teodoro-Martinez, D.S.; Barbosa, G.N.O.; Vaz, B.G.;
Silva, I.S.; Garcia, J.S.; T�otola, M.R.; Eberlin, M.N.; Grossman, M.;
Alves, O.L.; Durrant, L.R. Production and structural characterization
of surfactin (C14/Leu7) produced by Bacillus subtilis isolateLSFM-
05 grown on raw glycerol from the biodiesel industry. Process Bio-
chem. 2011, 46(10), 1951–1957.
[30] Kowall, M.; Vater, J.; Kluge, B.; Bein, B.T.; Franke, P.; Ziessow, D.
Separation and characterization of surfactin isoforms produced by
Bacillus subtilis OKB 105. J. Colloid Interface Sci. 1998, 204(1), 1–8.
[31] Lima, T.M.S.; Proc�opio L.C.; Brand~ao F.D.; Carvalho A.M.X.; T�otola
M.R.; Borges A.C. Biodegradability of bacterial surfactants. Biodegra-
dation 2011, 22(3), 585–592.
[32] Bondarenko, O.; Rahman, P.; Rahman, T.; Kahru, A.; Ivask, A.
Effects of rhamnolipids from Pseudomonas aeruginosa DS10-129 on
luminescent bacteria: Toxicity and modulation of cadmium bioavail-
ability. Microb. Ecol. 2010, 59(3), 588–600.
180 D. W. F. DE OLIVEIRA ET AL.
http://orcid.org/0000-0002-0866-9708
http://www.sigmaaldrich.com/catalog/product/sigma/s3523?lang=es&region=ES
http://www.sigmaaldrich.com/catalog/product/sigma/s3523?lang=es&region=ES
http://www.sigmaaldrich.com/catalog/product/sigma/s3523?lang=es&region=ES
http://www.sigmaaldrich.com/catalog/product/sigma/s3523?lang=es&region=ES
[33] Jurado, E.; Fern�andez-Serrano, M.; Olea, J.N.; Lechuga, M.; Jim�enez,
J.L.; R�ıos, F. Acute toxicity of alkylpolyglucosides to Vibrio fischeri,
Daphnia magna and microalgae: a comparative study. Bull. Environ.
Contam. Toxicol. 2012, 88(2), 290–295.
[34] Piętka-Ottlik, M.; Frąckowiak, R.; Maliszewska, I.; Ko»wzan, B.;
Wilk, K.A. Ecotoxicity and biodegradability of antielectrostatic
dicephalic cationic surfactants. Chemosphere 2012, 89(9), 1103–
1111.
[35] Francius, G.; Dufour, S.; Deleu, M.; Paquot, M.; Mingeot Leclercq, M.
P.; Dufr�ene, Y.F. Nanoscale membrane activity of surfactins: influ-
ence of geometry, charge and hydrophobicity. Biochim. Biophys.
Acta. 2008, 1778(10), 2058–2068.
[36] Sung, W.S.; Lee, D.G. The combination effect of Korean red ginseng
saponins with kanamycin and cefotaxime against methicillin-resistant
Staphylococcus aureus. Biol. Pharm. Bull. 2008, 31(8), 1614–1617.
[37] Avato, P.; Bucci, R.; Tava, A.; Vitali, C.; Rosato, A.; Bialy, Z.; Jurzysta,
M. Antimicrobial activity of saponins from Medicago sp.: structure–
activity relationship. Phytother. Res. 2006, 20(6), 454–457.
[38] El Izzi, A.; Benie, T.; Thieulant, M.L.; Le Men-Oliver, L.; Duval, J.
Stimulation of LH release from cultured pituitary cells by saponins
of Petersianthus macrocarpus: a permeabilising effect. Planta Med.
1992, 58(3), 229–233.
[39] Authi, K.S.; Rao, G.H.R.; Evenden, B.J.; Crawford, N. Action of gua-
nosine 5�-(beta-thio) diphosphate on thrombin-induced activation
and calcium mobilization in saponin-permeabilized and intact
human platelets. Biochem. J. 1988, 255(3), 885–894.
[40] Villegas-Navarro, A.; Romero Gonz�alez, M.C.; Lopez, E.R.; Aguilar,
R.D.; Marcal, W.S. Evaluation of Daphnia magna as an indicator of
toxicity and treatment efficacy of textile wastewaters. Environ. Int.
1999, 25(5), 619–624.
[41] Hodges, G.; Roberts, D.W.; Marshall, S.J.; Dearden, J.C. The aquatic
toxicity of anionic surfactants to Daphnia magna: a comparative
QSAR study of linear alkylbenzene sulphonates and ester sulpho-
nates. Chemosphere 2006, 63(9), 1443–1450.
[42] Ribosa, I.; Garcia, M.T.; Sanchez Leal, J.; Gonzalez, J.J. Photobacte-
rium phosphoreum test data of non-ionic surfactants. Toxicol. Envi-
ron. Chem. 1993, 39(3–4), 237–241.
[43] P�erez, L.; Pinazo, A.; Teresa Garc�ıa, M.; Lozano, M.; Manresa, A.;
Angelet, M., Vinardell, M.P.; Mijans, M.; Pons, R.; Rosa Infante, M.
Cationic surfactants from lysine: synthesis, micellization and biologi-
cal evaluation, Eur. J. Med. Chem. 2009, 44(5), 1884–1892.
[44] Blas�e, C.; Vasseys, P. Algal Microplate Toxicity Test. In Small-scale
Freshwater Toxicity Investigations; Blaise, C.; F�erard, J.F., Eds.;
Springer: Berlin, 2005; 137–179.
[45] Yamane, A.N.; Okada, M.; Sudo, R. The growth inhibition of plank-
tonic algae due to surfactants used in washing agents. Water Res.
1984, 18(9), 1101–1105.
[46] Cowan-Ellsberry, C.; Belanger, S.; Dorn, P.; Dyer, S.; McAvoy, D.;
Sanderson, H., Versteeg, D.; Ferrer, D.; Stanton, K. Environmental
safety of the use of major surfactants classes in North America. Crit.
Rev. Environ. Sci. Technol. 2014, 44(17), 1893–1993.
[47] Steger-Hartmann, T.; L€ange, R.; Schweinfurth, H. Environmental risk
assessment for the widely used iodinated X-ray contrast agent iopro-
mide (Ultravist). Ecotoxicol. Environ. Saf. 1999, 42(3), 274–281.
[48] Jurado, E.; Fern�andez-Serrano, M.; R�ıos, F.; Lechuga, M. Aerobic biodeg-
radation of surfactants. In Agricultural and biological sciences, Biodegra-
dation-life of science; Chamy, R., Ed.; InTech: Croatia, 2013; 63–79.
[49] Singh, K.L.; Kumar, A.; Kumar, A. Short communication: Bacillus
cereus capable of degrading SDS shows growth with a variety of
detergents. World J. Microbiol. Biotechnol. 1998, 14(5), 777–779.
[50] Jurado, E.; Fern�andez-Serrano, M.; Camacho, F.; N�u~nez-Olea, J.;
Lechuga, M.; Luz�on, G. Development and application of kinetic
models for the primary biodegradation of non-ionic surfactants.
Chem. Eng. J. 2009, 150(2–3), 440–446.
[51] Hirata, Y.; Ryu, M.; Oda, Y.; Igarashi, K.; Nagatsuka, A.; Furuta, T;,
Sugiura, M. Novel characteristics of sophorolipids, yeasts glycolipid
biosurfactants, as biodegradable low-foaming surfactants. J. Biosc.
Bioeng. 2009, 108(2), 142–146.
JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART A 181
	Abstract
	Introduction
	Material and methods
	Microorganism
	Culture medium
	Inoculum
	Biosurfactant production
	Glucose concentration
	Emulsification index
	Interfacial properties
	Structural characterization
	Acute toxicity tests
	Ready biodegradability tests
	Results and discussion
	Biosurfactant production and characterization
	Interfacial properties of the biosurfactant from B. subtilis ICA56
	Chemical structure of the biosurfactant produced by ICA56
	Toxicity of the biosurfactant produced by B. subtilis ICA 56 to aquatic organisms
	Acute toxicity test with Vibrio fischeri
	Acute toxicity test with Daphnia magna
	Toxicity test with the unicellular alga Selenastrum capricornutum
	Biodegradability assays
	Biodegradation by Pseudomonas putida
	Biodegradation of surfactin by activated sludge microorganisms
	Conclusions
	Funding
	References