CO2_2016 - page 55

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Chimica Oggi - Chemistry Today
- vol. 34(2) March/April 2016
frequently detected in effluent waters.
2. Modelling the degradation kinetics of both
compounds under laboratory conditions.
3. Detoxification process evaluation by a biomodel with
zebrafish embryos.
EXPERIMENTAL
Sewage water collection
Water samples were collected from urban WWTP. The main
supply is wastewater from two populations included in
Community of Madrid. This plant pours water to a stream
from the secondary treatment and purifies also through a
tertiary treatment to the required quality for use as irrigation
water. We collected three types of samples: riverbed water,
sample after secondary treatment (just before pouring
the stream) and water after completing tertiary treatment
(irrigation water for agricultural use). Samples were
refrigerated at 4 ºC until arrival at the laboratory.
Isolation and molecular identification of degrading
bacteria
Three cultures were prepared from each sample (final
volume 50 mL). 1 mL of sample was inoculated into
BHB (Bushnell Haas Broth) culture medium containing
Tween-80 (1%) as surfactant and carbamazepine (0.25
ppm) or naproxen (1.25 ppm) as pollutant. Tween-80 is
a surfactant, which enhances the solubility of PPCP´s. A
volume of 100 µL was plated in Petri dishes of each water
sample with LB agar + glucose and was incubated for
48 hours at 25°C in darkness. At least five colonies were
isolated from each plate per water sample.
In order to obtain biomass all that isolated colonies were
replated under the same conditions in individual plates.
A liquid culture medium in BHB with Tween-80 (1%) was
prepared from each colony biomass using carbamazepine
(0.125 ppm) or naproxen (0.625 ppm) as carbon sources.
Optical density evolution of 27 isolated colonies from water
samples over 12 days was studied. Liquid cultures that
showed cell growth (increment of the cell density), were
refreshed with liquid media to avoid that nutrients were
an inhibition factor of the cell growth or biodegradation
process. In order to select the most effective degrading
colony, those that showed greatest cell growth were
cultured again in a liquid media with PPCPs as sole carbon
source and followed by a new track over 19 days under the
same conditions mentioned above.
All incubations were performed on an orbital shaker
(New Brunswich Scientific, New Jersey USA) at 200
rpm and 25°C in darkness to avoid possible influence
of light on chemicals stability and contamination by
other microorganisms. The colony that showed the best
growth (C10) was obtained from the effluent treatment
plant discharge directly to the stream (after receiving
secondary treatment).
Finally, strain C10 was identifyed by extracting DNA
from the colony C10 and subsequent amplification and
sequencing as described in Molina et al., 2009 (22).
Naproxen and carbamazepine degradation kinetics
1 mL of a dilution in which was calculated the most
probable number (MPN) was incubated in a final
volume of 30 mL with a carbamazepine and naproxen
energy and environmental costs are the advantages of
bioremediation (11-13). Therefore, a promising perspective
consists in the application of these techniques where
microorganisms, microbiological aggregates and
commercial biological agents are involved. Nevertheless,
there are few research and development focused
on encouraging the improvement of bioremediation
techniques as compared with physicochemical projects
(Figure 1).
As a result, isolation and identification of new consortia
or isolates to degrade these compounds and adequate
ecotoxicological monitoring may have important
implications for the WWTP (Wastewater treatment plants).
Direct applications to improve the quality of effluents
could develop a management plan enabling them to
comply with the European Water Framework Directive and
future legislation.
Naproxen is one of the polar acidic pharmaceuticals most
used among human populations as an anti-inflamatory
and antipiyretic drug without prescription in several
countries (14). Naproxen mainly enter in aquatic systems
through municipal WWTP. Although it may be degraded
by indigenous communities (15), naproxen is one of the
most commonly detected drugs in European surface
waters included drinking water (16) due to factors such as
present microbial communities or environmental factors
such as seasonality (17). This compound can hamper
or inhibit microbial activities and reduce biodiversity of
natural microbial communities (18). Carbamazepine
has been detected in wastewater effluent. Field and
laboratoty studies have been conducted to understand
the accumulation of carbamazepine in food plants grown
in soil treated with sludge. Prosser et al. (19) report that
the accumulation of carbamazepine into plants grown in
soil amended with biosolids poses minimal risk to human
health according to the approach. However, the effect of
these emerging contaminant in more complex biological
models is little known.
On the other hand, zebrafish (
Danio rerio
) has been
extensively studied and well described for environmental
toxicity studies. Molecular biology and genetics
have recently been used to elucidate the underlying
mechanisms of toxicity in zebrafish and to predict effects
in mammals (20, 21).
The objectives of the present study are:
1. isolation and molecular identification of a microbial
strain capable of degrading naproxen (analgesic)
and/or carbamazepine (anticonvulsant) which are
Figure 1.
Number of publications related to pollutants
bioremediation techniques (brown) and physico-chemical
techniques (blue) since 2003.
1...,45,46,47,48,49,50,51,52,53,54 56,57,58,59,60,61,62,63,64,65,...68
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