CO2_2016 - page 56

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Chimica Oggi - Chemistry Today
- vol. 34(2) March/April 2016
of 48 h), growth (72 h of development) and behavior
(development of 144 h).
Modelling the PPCPs
metabolic kinetics
One of our goals is to model
the kinetics of the bacteria-
drugs reactions, so we will be
able to predict the outcome
of future experiments. In
order to extract the reaction
rates from the experimental
data, we have used the
method suggested in (30) to
optimise the Picard integral
operator associated to first
order differential equations.
Our very preliminary results
are plotted in Figure 2
as solid lines. They are in
good agreement with the
experimental data (also
plotted in the same Figure
2, with their corresponding
error bars), revealing
the behaviour of the
concentrations and bacteria
population that we discuss in
the following section.
RESULTS AND DISCUSSION
Naproxen was completely
removed by C10 strain
molecularly identified
as
Serratia
sp. within 7
days of culture. However,
carbamazepine
concentration remained constant until the end of analysis
(Figure 2).
To our knowledge this was the first time that a strain of
Serratia
is able to degrade naproxen. Other works described
the PPCP´s degradation (ibuprofen and naproxen) by
Pseudomanas putida
(23). Moreover, both river native
communities (17) and bacteria from air sludge reactors (24)
were able to easily degrade naproxen as well. The Gram-
positive strain
Planococcus
sp. removed approximately 30%
of naproxen after 35 days of incubation in monosubstrate
culture. Under cometabolic conditions, with glucose or
phenol as a growth substrate, the degradation efficiency
increased (25). In our experimental design where Tween-80
is added as surfactant, naproxen degradation is favoured
because this surfactant is also used as carbon source (11).
Molecules with more complex structures such as
carbamazepine are more difficult to remove with
bioremediation techniques (26). However, Rodríguez-
Rodríguez et al. (27) and Marco Urrea et al. (28) published
that
Trametes versicolor
degraded both naproxen and
carbamazepine (50-90%).
It is known that bioassay models are sensitive to the
presence of naproxen. Even at low concentrations if
mortality is not caused at least disturbance mobility is
concentration of 0.75 and 0.45 ppm, respectively. Cell
growth was monitored by optical density at 600 nm and
the pH of the medium (data
not shown). In this case,
triplicate samples of each
culture were taken over
time until a total of 21 days.
Samples were centrifuged
at 13000 rpm and 21°C for
10 min, in order to remove
cellular material. The
supernatant was used as a
target and the estimated
value was corrected from an
aliquot of the same volume
without centrifugation.
An extraction method using
chloroform was developed
to assess PPCP´s content in
the samples. The incubation
medium (30 mL) was
collected and naproxen
and carbamazepine were
extracted in a separatory
funnel with 3x5 mL of
chloroform. The 3 organic
phases were collected,
pooled, evaporated and
resuspended in 2 mL of
acetonitrile (ACN) and then
filtered through a 0.2 µm
cellulose membrane.
Degradation process
was analyzed by high
performance liquid
chromatography (HPLC)
on a reverse phase liquid
chromatograph equipped
with a Shimadzu UV-visible
detector and Phenomenex
®
Luna
®
C18 (2) (7.5 cm x 4.6
mm, 3μm) column. Analyses were performed in isocratic
mode, using a mobile phase acetonitrile:acid water (pH
4.0) 40:60 (v/v). The injection volume was 10 μL at a flow
rate of 0.5 ml/min. Both naproxen and carbamazepine
quantification was performed measuring characteristic
peaks areas of each compound using the corresponding
calibration standards (carbamazepine and naproxen
analytical standards, ≥ 99.0% HPLC, supplied by Sigma-
Aldrich).
Toxicological evaluation
A toxicological test with a model zebrafish embryo-larval
(
Brachydanio rerio
) was used to assess the toxicity of
the resulting samples and subsequently the treatment
efficiency. Water samples were collected at different
stages: an initial one before PPCP´s degradation by
microbial strain selected, 1-2 intermediate stages and final
stage of pollutants degradation. All water samples were
filtered to remove microbial fraction before exhibition to
zebrafish embryos.
Water samples toxicity with zebrafish embryo-larval model
was assessed by mortality (every 24 h of development),
malformations (24 h, 48 h and 72 h of development),
sublethal alterations such as cardiotoxicity (development
Figure 2.
Naproxen (green), carbamazepine (red) degradation
and
Serratia
concentration
(
cel ml
-1
) during analysis time.
Figure 3.
Detailed image of the chorion embryos. A) Control. B)
Altered chorion as a result of treatment.
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