Tesis de Carla Escapa Santos PDF

BIORREMEDIACIÓN DE AGUAS CONTAMINADAS CON NUTRIENTES Y FÁRMACOS MEDIANTE MICROALGAS
BIORREMEDIACIÓN DE AGUAS CONTAMINADAS

CON NUTRIENTES Y FÁRMACOS
MEDIANTE MICROALGAS
Departamento de Química y Física Aplicadas
Instituto de Medio Ambiente, Recursos Naturales y Biodiversidad
Universidad de León
Carla Escapa Santos

Carla
Escapa León, 2017
Santos

UNIVERSIDAD DE LEÓN
INSTITUTO DE MEDIO AMBIENTE, RECURSOS NATURALES Y
BIODIVERSIDAD
DEPARTAMENTO DE QUÍMICA Y FÍSICA APLICADAS

TESIS DOCTORAL
BIORREMEDIACIÓN DE AGUAS CONTAMINADAS CON

NUTRIENTES Y FÁRMACOS MEDIANTE MICROALGAS

Bioremediation of contaminated waters

with nutrients and pharmaceuticals by microalgae

Dirigida por: Presentada por
Dra. Ana Isabel García Pérez Carla Escapa Santos
Dra. Marta Otero Cabero para optar al grado de Doctor

León, 2017
Tesis Doctoral por compendio de publicaciones
Nutrients and pharmaceuticals removal from wastewater by culture and harvesting of

Chlorella sorokiniana
Escapa C, Coimbra R.N, Paniagua S, García A.I, Otero M
Bioresource Technology, 185 (2015): 276‐284
Paracetamol and salicylic acid removal from contaminated water by microalgae
Journal of Environmental Management, (2016) doi: 10.1016/j.jenvman.2016.06.051
Comparison of the culture and harvesting of Chlorella vulgaris and Tetradesmus obliquus
for the removal of pharmaceuticals from water
Journal of Applied Phycology, (2016) doi: 10.1007/s10811‐016‐1010‐5
Comparative assessment of diclofenac removal from water by different microalgae strains
Algal Research, 18 (2016): 127‐134
Ability of microalgae bioremediation systems to reduce the toxicity of pharmaceuticals

contaminated waters. Toxicity assessment with zebrafish (Danio rerio) embryo bioassays
Escapa C, Torres T, Neuparth T, Santos M.M, García A.I, Otero M
Enviado a Water Reseach (marzo de 2017)

Agradecimientos
Quiero expresar mi agradecimiento más sincero a mi directora de tesis, Ana Isabel García,
por haber confiado en mí y ofrecerme la oportunidad de realizar esta tesis; a mi directora
Marta Otero, por transmitirme su pasión por la investigación y por su incondicional apoyo; y a
todas aquellas personas que bien desde el ámbito profesional o desde el personal han hecho
posible el desarrollo de esta investigación.
A todos ellos, gracias.

Financiación
Esta tesis se ha podido desarrollar gracias a la concesión de una beca del Programa de
Formación de Profesorado Universitario (FPU) del Ministerio de Educación, Cultura y Deporte
(FPU12/03073). Dentro del desarrollo de la beca se realizó una estancia en el centro de
investigación Interdisciplinary Centre of Marine and Environmental Research en el laboratorio del
Dr. Miguel Alberto Fernandes Machado e Santos (Porto, Portugal). Además, se ha contado con la
financiación de la Universidad de León a través de la concesión del proyecto MICROTRAT
(proyecto UXXI2016/00128).
Tabla de contenido
Tabla de contenido ............................................................................................................................. IX
Resumen .............................................................................................................................................XIII
Abstract ............................................................................................................................................. XV
Capítulo 1. Introducción general ......................................................................................................... 1
1.1. Justificación de la unidad temática ................................................................................................. 3
1.2. Presentación de las publicaciones ................................................................................................... 4
Capítulo 2. Antecedentes ................................................................................................................... 7
2.1. La problemática de las aguas residuales ......................................................................................... 9
2.2. Los contaminantes emergentes ...................................................................................................... 10
2.3. Los fármacos como contaminantes emergentes ............................................................................. 13
2.4. Efectos y riesgos de la presencia de fármacos en el medio ambiente ............................................ 16
2.5. Sistemas de tratamiento en la eliminación de contaminantes emergentes del agua .................... 18
2.5.1. Sistemas convencionales ......................................................................................................... 18
2.5.2. Tratamientos avanzados ......................................................................................................... 20
2.5.3. Tratamientos alternativos ....................................................................................................... 24
2.6. Utilización de las microalgas en el tratamiento de aguas residuales .............................................. 26
2.6.1. Implementación en los sistemas de tratamiento de aguas residuales convencionales .......... 28
2.6.2. Mecanismos de eliminación de nitrógeno y fósforo ................................................................ 31
2.6.3. Mecanismos de eliminación de contaminantes emergentes .................................................. 32
2.6.4. Separación de la biomasa ....................................................................................................... 33
2.7. Referencias bibliográficas ................................................................................................................ 34
Capítulo 3. Objetivos .......................................................................................................................... 51
3.1. Objetivos .......................................................................................................................................... 53
IX
Capítulo 4. Artículo I ........................................................................................................................... 55
Nutrients and pharmaceuticals removal from wastewater by culture and harvesting
of Chlorella sorokiniana .......................................................................................................................... 57
Capítulo 5. Artículo II .......................................................................................................................... 71
Paracetamol and salicylic acid removal from contaminated water by microalgae ................................ 73
Capítulo 6. Artículo III ......................................................................................................................... 87
Comparison of the culture and harvesting of Chlorella vulgaris and Tetradesmus obliquus
for the removal of pharmaceuticals from water .................................................................................... 89
Capítulo 7. Artículo IV ......................................................................................................................... 105
Comparative assessment of diclofenac removal from water by different microalgae strains ............... 107
Capítulo 8. Artículo V .......................................................................................................................... 115
Ability of microalgae bioremediation systems to reduce the toxicity of pharmaceuticals
contaminated waters. Toxicity assessment with zebrafish (Danio rerio) embryo bioassays ................. 117
Capítulo 9. Resumen de resultados y discusión ................................................................................... 135
9.1. Crecimiento de las microalgas durante la biorremediación de aguas contaminadas
con fármacos ..................................................................................................................................... 137
9.1.1. Efectos del paracetamol sobre el crecimiento ......................................................................... 137
9.1.2. Efectos del ácido salicílico sobre el crecimiento ...................................................................... 138
9.1.3. Efectos del diclofenaco sobre el crecimiento ........................................................................... 139
9.2. Eliminación de nutrientes durante la biorremediación de aguas contaminadas
con fármacos ..................................................................................................................................... 142
9.2.1. Eliminación de nutrientes bajo la presencia de paracetamol .................................................. 142
9.2.2. Eliminación de nutrientes bajo la presencia de ácido salicílico ............................................... 144
9.2.3. Eliminación de nutrientes bajo la presencia de diclofenaco .................................................... 146
X
9.3. Eliminación de fármacos durante la biorremediación de aguas contaminadas .............................. 149
9.3.1. Eliminación de paracetamol .................................................................................................... 150
9.3.2. Eliminación de ácido salicílico ................................................................................................. 151
9.3.3. Eliminación de diclofenaco ...................................................................................................... 152
9.3.4. Eliminación de altas concentraciones relativas de paracetamol y ácido salicílico .................. 156
9.4. Coagulación‐floculación como sistema de separación de la biomasa de microalgas
del agua tratada ................................................................................................................................ 158
9.4.1. Coagulación‐floculación mediante sales metálicas ................................................................. 158
9.4.2. Coagulación‐floculación mediante polielectrolitos sintéticos ................................................. 161
9.4.3. Coagulación‐floculación mediante chitosan ........................................................................... 164
9.5. Efectos sobre el desarrollo embrionario del pez cebra (Danio rerio) para evaluar
la toxicidad de los efluentes .............................................................................................................. 166
9.5.1. Bioensayos con paracetamol .................................................................................................. 167
9.5.2. Bioensayos con ácido salicílico ................................................................................................ 169
9.5.3. Bioensayos con diclofenaco ..................................................................................................... 170
9.6. Referencias bibliográficas ................................................................................................................ 172
Capítulo 10. Conclusiones ....................................................................................................................177
10.1. Conclusiones .................................................................................................................................. 179
10.2. Conclusions .................................................................................................................................... 181
XI
Resumen
La presente tesis se centra en el estudio de la aplicación de las microalgas como sistema de
biorremediación de aguas contaminadas por nutrientes y fármacos. Para ello se determinó la
influencia de la presencia de fármacos sobre el crecimiento de las especies Chlorella sorokiniana,
Chlorella vulgaris y Scenedesmus obliquus. Estas especies fueron cultivadas en fotobiorreactores
tipo columna de burbujeo bajo condiciones controladas en modo discontinuo y semicontinuo.
Tanto las curvas como los parámetros de crecimiento correspondientes mostraron que la
presencia de fármacos no afectó negativamente al desarrollo de los cultivos, presentando, en la
mayoría de los casos, un incremento significativo de la concentración de biomasa.
La eficiencia en la eliminación de nitratos y fosfatos de las especies de microalgas antes

referidas se evaluó en función de los valores alcanzados al final del cultivo discontinuo y durante
el estado estacionario del cultivo semicontinuo. La presencia de los fármacos estudiados no
interfirió con la eliminación de nutrientes en ningún caso. Al final del cultivo discontinuo se
alcanzó en todos los casos una eliminación casi completa de ambos nutrientes. Sin embargo,
durante el estado estacionario del cultivo semicontinuo, fue S. obliquus la especie que alcanzó las
mayores eficiencias de eliminación de nutrientes.
Para abordar la eficiencia de eliminación de los fármacos paracetamol, ácido salicílico y

diclofenaco mediante las microalgas aquí presentadas, se estudió la cinética de eliminación
durante el cultivo discontinuo así como la tasa de eliminación durante el estado estacionario del
cultivo semicontinuo. Los resultados obtenidos indican la viabilidad de la utilización de las
microalgas aquí estudiadas como sistema de biorremediación. Comparando los tres fármacos, el
ácido salicílico fue el fármaco eliminado con una mayor eficiencia, mostrando C. sorokiniana y
S. obliquus los mejores resultados. Contrariamente, el paracetamol fue el fármaco que se eliminó
con una peor eficiencia. Además, ha quedado demostrada no solo la resistencia, sino también la
capacidad de eliminación de altas concentraciones relativas de paracetamol y ácido salicílico
mediante C. sorokiniana.
Con el objetivo de proponer una opción para la separación de la biomasa del efluente
tratado, se evaluaron las eficiencias de diferentes agentes floculantes y dosis en la recuperación
de la biomasa de las especies C. sorokiniana, C. vulgaris y S. obliquus. Los floculantes estudiados
fueron sales metálicas (AlCl3 y FeCl3), polielectrolitos sintéticos (CH‐35 y CH‐15) y un floculante
orgánico (chitosan). A pesar de que los polielectrolitos permitieron recuperaciones de la biomasa
prácticamente inmediatas (1 minuto), la viscosidad del flóculo generado apuntó hacia las sales
metálicas como la mejor opción.
XIII

Finalmente, con el fin de evaluar la capacidad de las microalgas en la disminución de la

toxicidad de los efluentes tratados, se realizaron bioensayos de desarrollo embrionario del pez
cebra (Danio rerio), estudiando los efectos producidos sobre la mortalidad y las anomalías
morfológicas en diferentes estados de desarrollo. La eficiencia de las microalgas en la disminución
de la toxicidad se evaluó comparando dichos efectos con aquellos producidos por la dosis inicial
del fármaco. Así se pudo comprobar que los sistemas de biorremediación mediante C. sorokiniana,
C. vulgaris y S. obliquus no solo fueron capaces de reducir la concentración de fármacos en el agua
tratada, sino también su toxicidad. Por lo tanto, la eliminación de paracetamol, ácido salicílico y
diclofenaco mediante estas especies de microalgas no dio lugar a productos de transformación
tóxicos, lo que es de suma importancia de cara a su aplicación en la biorremediación de aguas
contaminadas con fármacos.
XIV

Abstract
The present thesis is focused on the study of the ability of microalgae as bioremediation
system of contaminated waters by nutrients and pharmaceuticals. For this purpose, the influence
of pharmaceuticals in the growth of the strains Chlorella sorokiniana, Chlorella vulgaris and
Scenedesmus obliquus was determined. These microalgae strains were cultivated in bubbling
column photobioreactors under controlled conditions in batch and semicontinuous mode. Growth
curves and parameters pointed out that the culture was not negatively affected by the presence of
pharmaceuticals, showing in most of the cases a significant increase of the biomass concentration.
The efficiency in the removal of nitrates and phosphates by the microalgae strains
aforementioned was evaluated according to the values obtained at the end of the batch culture
and at the steady state of the semicontinuous culture. The presence of the studied
pharmaceuticals did not interfere in the removal of nutrients in any case. At the end of the batch
culture, the removal of nutrients was nearly complete in all cases. However, at the steady state of
the semicontinuous culture, S. obliquus was the strain that reached the highest efficiencies in the
removal of nutrients.
To address the removal efficiency of the pharmaceuticals paracetamol, salicylic acid and
diclofenac by the microalgae here considered, the removal kinetics during batch culture as well as
the removal rate during the steady state of the semicontinuous culture were determined. The
results obtained pointed to the feasibility of the microalgae here studied as bioremediation
system. Among the three pharmaceuticals, salicylic acid was the most efficiently removed, and the
best results were obtained with C. sorokininana and S. obliquus. Contrary, paracetamol was the
poorest efficiently removed. Furthermore, the robustness and the removal efficiency of
C. sorokiniana under relative high concentrations of paracetamol were proved.
After treatment, aiming to find out a way to separate the microalgae biomass from the
treated effluent, the efficiencies of different flocculants and doses were evaluated in the recovery
of the biomass of the strains C. sorokiniana, C. vulgaris y S. obliquus. Metal salts (AlCl3 and FeCl3),
synthetic polyelectrolytes (CH‐35 and CH‐15) and an organic flocculant (chitosan) were assessed.
In spite of polyelectrolytes being able to recover biomass almost immediately (1 minute), the floc
viscosity pointed to metal salts as the best option.
XV

Finally, in order to assess the ability of microalgae to reduce the toxicity of the treated
effluents, embryonic development bioassays of zebrafish (Danio rerio) were performed, studying
the effects over the mortality and morphological abnormalities in different developmental stages.
The microalgae efficiency in the toxicity reduction was evaluated by comparing these effects with
those produced by the initial concentration of pharmaceuticals. In this way, it was verified that
bioremediation systems by C. sorokiniana, C. vulgaris and S. obliquus not only reduced the
pharmaceutical concentration but also the toxicity of the effluents. Therefore, the removal of
paracetamol, salicylic acid and diclofenac by these microalgae strains did not result in toxic
transformation products, which is utmost important for their application in the bioremediation of
pharmaceutical contaminated waters.
XVI

Capítulo 1

Introducción general

1.1 JUSTIFICACIÓN DE LA UNIDAD TEMÁTICA
En los últimos años se ha despertado una gran preocupación social y científica tras la
detección de nuevos contaminantes en diferentes matrices ambientales de los que se desconocen
los potenciales efectos adversos que pueden generar sobre la salud humana y el medio ambiente.
Estos contaminantes emergentes incluyen una amplia variedad de productos de uso diario con
aplicaciones tanto industriales como domésticas. Entre ellos, los fármacos han recibido una especial
atención debido a que su presencia en el medio ambiente puede ocasionar respuestas fisiológicas
en organismos para los que no fueron destinados. El hecho de que hayan sido detectados en aguas
superficiales, subterráneas, marinas e incluso en aguas potables, está relacionado con la
incapacidad de las estaciones de depuración de aguas residuales (EDARs) para eliminar estos
compuestos. A esta ineficaz eliminación, se suma la falta de regulación en cuanto a las
concentraciones máximas admisibles en el medio ambiente para la gran mayoría de los
contaminantes emergentes.
La preocupación por la presencia de productos farmacéuticos en el medio acuático ha llevado

a la reciente consideración de estos compuestos en la normativa europea en la Directiva Marco del
Agua (2000/60/CE) . Fue en la Propuesta de la Comisión Europea de 31 de enero de 2012 , cuando se

propuso la inclusión en la lista de sustancias prioritarias de tres fármacos, diclofenaco, 17‐beta‐
estradiol (E2) y 17‐alfa‐etinilestradiol (EE2). Finalmente, la Decisión de la UE 2015/495 , incluyó

estos compuestos, junto con la estrona (E1) y tres antibióticos (azitromicina, claritromicina y
eritromicina), en la primera lista de observación, formada por aquellas sustancias que deben
vigilarse en todos los Estados miembros para respaldar futuras revisiones de la lista de sustancias
prioritarias.
Directiva 2000/60/CE del Parlamento Europeo y del Consejo, de 23 de octubre de 2000, por la que se establece un
marco comunitario de actuación en el ámbito de la política de aguas. Diario Oficial de las Comunidades Europeas,
22‐12‐2000, L327/1‐L327‐73.
Propuesta COM(2011) 876 final de Directiva del Parlamento Europeo y del Consejo, de 31 de enero de 2012, por la
que se modifican las Directivas 2000/60/CE y 2008/105/CE en cuanto a las sustancias prioritarias en el ámbito de
la política de aguas. https://ec.europa.eu/transparency/regdoc/rep/1/2011/ES/1‐2011‐876‐ES‐F1‐1.pdf.
Decisión de la Ejecución (UE) 2015/495 de la Comisión, de 20 de marzo de 2015, por la que se establece una lista
de observación de sustancias a efectos de seguimiento a nivel de la Unión en el ámbito de la política de aguas, de
conformidad con la Directiva 2008/105/CE del Parlamento Europeo y del Consejo. Diario Oficial de la Unión
Europea, 24‐3‐2015, L78/40‐L78/42.
3
CAPÍTULO 1
Los sistemas biológicos han surgido como una alternativa sostenible y económicamente
rentable en el tratamiento de aguas residuales para la eliminación de contaminantes emergentes.
Entre ellos, la biorremediación con microalgas, a pesar de poco estudiada, ha sido presentada como
una opción prometedora. Las microalgas se caracterizan por tener altas eficiencias fotosintéticas,
altas tasas de crecimiento, una amplia adaptabilidad a diferentes condiciones de cultivo y una gran
capacidad para eliminar nutrientes inorgánicos del agua. La principal ventaja del uso de microalgas
en la eliminación de nutrientes en los sistemas convencionales de tratamiento del agua, es que
permite reciclar el nitrógeno y fósforo asimilado en la biomasa, además de originar un efluente con
elevada concentración de oxígeno disuelto. Sin embargo, a pesar de que la capacidad de las
microalgas en la eliminación de nutrientes del agua ya ha sido demostrada, el conocimiento sobre
la capacidad de eliminación de contaminantes emergentes, y más aún en fármacos, es un campo de
reciente investigación.
1.2. PRESENTACIÓN DE LAS PUBLICACIONES
La presente tesis es un compendio de cinco publicaciones en las que se estudian diferentes

aspectos sobre la aplicación de las microalgas en sistemas de biorremediación de aguas
contaminadas por nutrientes y fármacos.
En el Capítulo 4 (Artículo I) “Nutrients and pharmaceuticals removal from wastewater by

culture and harvesting of Chlorella sorokiniana”, se estudia la capacidad de C. sorokiniana para la
eliminación de paracetamol y ácido salicílico del agua. Además, se estudia la eliminación simultánea
de nitratos y fosfatos del medio. Finalmente, se propone la floculación como sistema de cosechado
de la biomasa, estudiando diferentes agentes coagulantes‐floculantes y dosis.
En el Capítulo 5 (Artículo II) “Paracetamol and salicylic acid removal from contaminated
water by microalgae” se evalúa la viabilidad de la microalga Chlorella sorokiniana, como sistema de
tratamiento para la eliminación de paracetamol y ácido salicílico de aguas con altas
concentraciones relativas de dichos fármacos.
En el Capítulo 6 (Artículo III) “Comparison of the culture and harvesting of Chlorella

vulgaris and Tetradesmus obliquus for the removal of pharmaceuticals from water” se comparan las
especies C. vulgaris y T. obliquus como sistema para la eliminación de paracetamol y ácido salicílico
del agua. Así mismo, se determina la influencia de la presencia de dichos fármacos en la eliminación
de nutrientes mediante microalgas. Finalmente, se comparan ambas especies respecto a su
capacidad de floculación con el objetivo de recuperar la biomasa.
4

En el Capítulo 8 (Artículo IV) “Comparative assessment of diclofenac removal from water

by different microalgae strains” se evalúa la eliminación de diclofenaco del agua mediante
microalgas. Además, considerando la aplicación de las microalgas en el tratamiento de aguas
residuales, se estudia la influencia de la presencia de este fármaco en la eliminación de nutrientes.
Para llevar a cabo este estudio, se seleccionaron las especies Chlorella sorokiniana, Chlorella
vulgaris y Scenedesmus obliquus, con el objetivo de comparar las eficiencias obtenidas por las
diferentes especies.
En el Capítulo 9 (Artículo V) “Ability of microalgae bioremediation systems to reduce the

toxicity of pharmaceuticals contaminated waters. Toxicity assessment with zebrafish (Danio rerio)
embryo bioassays” se evalúa la disminución de la toxicidad del efluente obtenido tras la
biorremediación de fármacos mediante microalgas. Los bioensayos sobre el desarrollo embrionario
de Danio rerio permiten evaluar los efectos generados por las concentraciones residuales de
diclofenaco, paracetamol y ácido salicílico tras la biorremediación con Chlorella sorokiniana,
Chlorella vulgaris y Scenedesmus obliquus; y compararlos con los efectos que producirían sin el
tratamiento mediante microalgas.
5

Capítulo 2
Antecedentes
Antecedentes
2.1. LA PROBLEMÁTICA DE LAS AGUAS RESIDUALES
El rápido desarrollo humano y económico, junto con el uso inadecuado del agua, han
generado un preocupante deterioro de la calidad del medio ambiente acuático. Durante décadas,
toneladas de sustancias biológicamente activas han sido vertidas sin reparar en las posibles
consecuencias. Las aguas residuales urbanas, industriales y las de origen agrícola o ganadero son
las principales vías de contaminación del medio acuático. Además, el incremento en el consumo
del agua y el descubrimiento de nuevos contaminantes deja patente la necesidad de investigar en
aquellas áreas que puedan contribuir a proteger la salud humana y el medio ambiente, además de
conseguir un uso sostenible del agua.
En la actualidad, las EDARs tienen que hacer frente por una parte, a una mayor
concentración de contaminantes convencionales y por otra parte, a la aparición de nuevos
contaminantes. La Directiva 98/15/CE de 27 de febrero, que modifica la Directiva 91/271/CE de 21
de mayo en su Anexo I, establece los requerimientos para las descargas de EDARs en áreas con
riesgo de eutrofización, estableciendo el límite en 2 mg l-1 la concentración de fósforo total y en
15 mg l-1 la concentración de nitrógeno total. Además, la Directiva 91/271/CE de 21 de mayo,
sobre el tratamiento de aguas residuales urbanas, establece los límites en Demanda Biológica de
Oxígeno (DBO5) en 25 mg l-1 O2, Demanda Química de Oxígeno (DQO) en 125 mg l-1 O2 y Sólidos
Suspendidos Totales (SST) en 35 mg l-1. Por otra parte, ciertos compuestos como son los
hidrocarburos aromáticos policíclicos, los plaguicidas y algunos metales han sido evaluados para
el cumplimiento de los criterios sanitarios de la calidad del agua de consumo humano en
cumplimiento de la Directiva 98/83/CE de 3 de noviembre. Otros, como es el caso de los
contaminantes emergentes se encuentran en reciente fase de control. Algunos de ellos han sido
incluidos en la lista de sustancias prioritarias (Directiva 2008/105/CE), otras se encuentran tan
solo en una fase de evaluación, formando parte de una lista de observación (Decisión de la
Ejecución (UE) 2015/495 de la Comisión).
A pesar de que progresivamente se han ido adoptando medidas legislativas para reducir la
contaminación del agua y los riesgos derivados, es necesario seguir investigando en todas
aquellas áreas que puedan contribuir a proteger la salud humana y la del medio ambiente.
9
CAPÍTULO 2
2.2. LOS CONTAMINANTES EMERGENTES
El reciente desarrollo de nuevos y más sensible métodos analíticos, ha permitido identificar

y cuantificar nuevos contaminantes en el agua, cuya presencia en el medio ambiente no es
necesariamente nueva. A pesar de que estos compuestos han sido detectados en concentraciones
de µg l-1 o ng l-1 en aguas naturales, se ha despertado una creciente preocupación social y científica
debido a los potenciales efectos adversos que estos contaminantes pueden generar sobre la salud
humana y el medio ambiente. Además, debido a su elevada producción y consumo, su
introducción en el medio ambiente es continua, lo hace que no necesiten ser persistentes para
encontrarse en el medio ambiente y ocasionar efectos negativos (Petrovic et al., 2003).
Entre los contaminantes emergentes se incluyen una amplia variedad de productos de uso
diario con aplicaciones tanto industriales como domésticas, como son compuestos farmacéuticos,
productos de cuidado e higiene personal, drogas de abuso, plaguicidas, aditivos industriales, etc.
(Tabla 2.1). Muchos de estos compuestos han sido detectados en las aguas residuales de plantas
de tratamiento y también en aguas superficiales, subterráneas, marinas e incluso en aguas
potables (Benotti et al., 2009; Bester et al., 2001; Bono-Blay et al., 2012; Brausch and Rand, 2011;
Deblonde and Cossu-Leguille, 2011; Kuster et al., 2008; Pal et al., 2010).
La presencia de estos contaminantes en aguas naturales está relacionada principalmente

con la incapacidad de las EDARs de eliminar estos compuestos y los metabolitos y/o productos de
degradación originados, que pueden ser incluso más tóxicos que los compuestos
originales (Petrie et al., 2015). A esta ineficaz eliminación, se suma la falta de regulación en cuanto
a las concentraciones máximas que se pueden verter en el medio ambiente para la gran mayoría
de los contaminantes emergentes.
A pesar de haberse registrado más de 10.000 sustancias potencialmente peligrosas en la

Unión Europea, en la actualidad tan solo 45 compuestos han sido identificados como prioritarios
por la Directiva Marco del Agua y recogidos en las Normas de Calidad Ambiental según la
Directiva 2008/105/CE, en la que se fijan las concentraciones máximas admisibles en el agua.
Otros, se encuentran actualmente en fase de evaluación para su posible inclusión en la lista de
sustancias prioritaria en futuras revisiones, formando parte actualmente de la primera lista de
observación.
10
Antecedentes
Tabla 2.1 Principales contaminantes emergentes (García-Navas, 2013).
Antibióticos, analgésicos, antiinflamatorios, antidepresivos,

Productos farmacéuticos ansiolíticos, reguladores de lípidos, β- bloqueadores, medios de
contraste para rayos X
Drogas de abuso Cocaínicos, anfetamínicos, cannabinoides, opiáceos, alucinógenos
Hormonas esteroideas Estradiol, estrona, estriol, dietilestilbestrol
Fragancias, nitropolicíclicos, macrocíclicos, parabenos,

Productos de cuidado personal
antimicrobianos
Agentes para la protección solar Benzofenona, metilbenzilideno
Repelentes N N-dietil-toluamida
Antisépticos Triclosán o clorofeno
Agentes de limpieza y detergentes Alquifenoles y carboxilados alquifenoles
Retardantes de llama Éteres polibromados
Agentes y aditivos industriales 2-cloroetilfosfato
Aditivos para la gasolina Éteres de aquilos o éter metil-t-butilo
Subproductos de la desinfección Bromoácidos, bromoacetonitrilos, bromatos
Otras sustancias Nicotina, cafeína
En las últimas décadas, muchas organizaciones de reconocida relevancia han tomado

conciencia del problema, teniendo entre sus líneas de investigación prioritarias la problemática
de los contaminantes emergentes, como la Organización Mundial para la Salud (OMS), la Unión
Europea (EU), el Programa de Naciones Unidas para el Medio Ambiente (OMS, PNUMA), la
Agencia para la Protección medioambiental de Estados Unidos (EPA) o el Programa Internacional
de Seguridad Química (IPCS). Por ello, la comunidad científica ha realizado un gran esfuerzo para
determinar la presencia y la concentración de los contaminantes emergentes en las complejas
matrices ambientales, para evaluar los posibles efectos en la salud y el medio ambiente y para
investigar sobre tratamientos alternativos de aguas residuales capaces de eliminar estos
11
CAPÍTULO 2
contaminantes de los vertidos. Prueba de ello es la publicación en los últimos años, de numerosos
artículos y revisiones bibliográficas en este área de investigación (de Wilt et al., 2016; García-
Rodríguez et al., 2014; Gupta et al., 2015; Luo et al., 2014; Matamoros et al., 2016; Norvill et al.,
2016; Tiedeken et al., 2017). La investigación sobre contaminantes emergentes pretende
promover medidas que permitan mejorar la calidad de las aguas, optimizar la explotación de los
recursos hídricos y proteger la salud del hombre y del medio ambiente. En este sentido, se pueden
distinguir tres líneas principales de investigación que se plantean a seguir.
El primer paso para dar respuesta a la problemática que plantean los contaminantes
emergentes ha sido desarrollar nuevos y más sensibles métodos analíticos que permitan la
determinación de estos contaminantes en las complejas matrices ambientales, tales como las
aguas superficiales y subterráneas, las aguas residuales y los suelos. La cromatografía líquida y
gaseosa acoplada a espectrometría de masas simple o en tándem, son las técnicas analíticas
actualmente utilizadas para este propósito, las cuales permiten detectar estos compuestos a muy
bajas concentraciones, del orden de partes por billón o partes por trillón. Además, es necesario el
desarrollo de protocolos para la toma de muestras y preparación previa al análisis. En el caso de
los fármacos, su utilización y consumo resulta en la excreción del propio fármaco inalterado junto
con subproductos del metabolismo, en proporciones muy variables según el fármaco, la dosis, el
individuo, etc. La identificación analítica de los subproductos de eliminación del metabolismo tras
el consumo del fármaco, resulta muy difícil debido a la escasez de patrones y a las técnicas de
identificación, lo que hace necesario disponer de protocolos que permitan su identificación,
además de determinar su toxicidad.
El siguiente paso, comprende la evaluación del destino y biodisponibilidad de estos

contaminantes en el medio ambiente, ya que es necesario conocer la concentración de estos
compuestos que llega a los tejidos sobre los que tienen efecto. Además, es necesario investigar
sobre el tipo de transformaciones que experimentan los contaminantes emergentes en los
procesos de depuración de aguas residuales así como en el propio medio ambiente acuático.
Así mismo, resulta imprescindible estudiar los efectos potenciales de estos compuestos sobre el
medio ambiente y la salud. Estos estudios de toxicidad deben comprender los metabolitos y
productos de degradación, además de los compuestos originales.
Por último, otra de las líneas de investigación prioritarias se centra en el estudio de

tratamientos de aguas residuales alternativos que permitan reducir su introducción en el medio
ambiente por esta vía, ya que en la actualidad constituye la principal fuente de los contaminantes
emergentes en el medio ambiente (Chaukura et al., 2016; Hwang et al., 2016; Karaolia et al., 2017;
Philippe et al., 2016; Rozas et al., 2016; Wang and Wang, 2017; Wang et al., 2016).
12
Antecedentes
2.3. LOS FÁRMACOS COMO CONTAMINANTES EMERGENTES
Entre los contaminantes emergentes, los que probablemente han suscitado una mayor
preocupación y estudio en los últimos años son los fármacos. El consumo de fármacos se está
incrementando en un 3-4% cada año, por lo que miles de toneladas de fármacos se vierten al agua
anualmente (Figura 2.1) (OECD, 2017).
Las primeras evidencias de la presencia de fármacos en el agua se remontan a los años 70

con la detección de ácido clofíbrico en las aguas residuales de EEUU, utilizado para disminuir los
niveles de lípidos en sangre. Sin embargo, no fue hasta principios de la década de los 90 cuando la
preocupación sobre los efectos en el medio ambiente y la salud tomó un mayor peso en la
sociedad, como demuestran los numerosos artículos publicados desde entonces (Halling-
Sorensen et al., 1998; Hom-Díaz et al., 2015; Sanderson et al., 2004; Villaescusa et al., 2011).
Actualmente, en la Unión Europea se han cifrado en 3.000 los distintos tipos de fármacos
disponibles en el mercado (Kümmerer, 2003).
60000
Dosis diaria/ 1000 habitantes día
50000
40000
30000
20000
10000
Figura 2.1 Tendencia en el consumo de fármacos de los países europeos pertenecientes a la OECD (adaptación de
Organisation for the Economyc Co-operation and Development, datos actualizados en octubre de 2016).
Con el objetivo de afrontar el riesgo que suponen estas sustancias, la Comisión Europea
propuso la inclusión de tres fármacos en la lista de sustancias prioritarias, siendo éstas el
antiinflamatorio diclofenaco y los estrógenos 17-β-estradiol (E2) y 17-α-etinilestradiol (EE2), a
través de la Propuesta Directiva (COM(2011)876 final). Sin embargo, esta propuesta fue rechazada
y, finalmente, mediante la Decisión de la Ejecución (UE) 2015/495 de la Comisión, estos fármacos
fueron incluidos, junto con otro estrógeno y tres antibióticos, en la primera lista de observación en
el contexto de la Directiva Marco del Agua (Directiva 2000/60/CE).
13
CAPÍTULO 2
La entrada de contaminantes de tipo farmacológico al medio acuático puede deberse a los

residuos derivados del uso terapéutico en medicina humana (doméstico u hospitalario), los
residuos derivados del uso terapéutico veterinario y los residuos generados en la industria
farmacéutica (Figura 2.2). A estos residuos de difícil control, se suma la inadecuada eliminación
de fármacos caducados o sobrantes a los residuos domésticos o directamente al agua.
Cada fármaco, una vez administrado, puede ser excretado sin sufrir ninguna
transformación, o bien ser metabolizado mediante reacciones de oxidación, reducción, hidrólisis y
alquilación, o bien formar conjugados más polares e hidrofílicos que son excretados por la orina o
la bilis (Heberer, 2002a). Los fármacos utilizados en medicina veterinaria pueden ser excretados
al suelo, o directamente a las aguas superficiales, por lo que su control es difícil. En el caso de la
ganadería intensiva, estos medicamentos son susceptibles de entrar al medio ambiente a través
del suelo en su uso agrícola mediante la incorporación de estiércol y como abono líquido. Por otra
parte, los medicamentos suministrados en las piscifactorías son, normalmente, liberados
directamente a las aguas superficiales (Halling-Sorensen et al., 1998). La mayoría de estos
compuestos y sus metabolitos son solubles en agua, en donde, entre el 55-80% de la cantidad
total suministrada es excretada a través de la orina (Heberer, 2002a).
Por otra parte, las aguas residuales procedentes de la industria farmacéutica se

caracterizan por presentar una gran variabilidad en cuanto a caudal y composición. Estos
parámetros van a depender del régimen de producción, la elaboración concreta que se esté
llevando a cabo, del tipo de actividad que esté generando el residuo, etc. Todas estas variables
hacen que la contaminación del efluente final pueda ser muy diversa y variable en el tiempo. El
volumen más importante se produce durante el lavado de los equipos al finalizar el proceso de
producción. Según la Directiva 91/271/CEE, los vertidos industriales que se hagan al sistema de
depuración de aguas residuales urbanas, deberán asegurar un tratamiento previo.
En función de las propiedades físico-químicas de los fármacos, metabolitos y/o productos

de degradación, y las características de los suelos, estas sustancias pueden llegar a alcanzar las
aguas subterráneas o bien quedar retenidas y acumularse en el suelo, pudiendo afectar al
ecosistema y a la salud humana a través de la cadena trófica.
14
Antecedentes
Figura 2.2 Principales vías de entrada de los fármacos al agua (fuente: elaboración propia).
Recientes estudios han demostrado que los sistemas de tratamiento convencionales

resultan ineficaces en la eliminación de fármacos de las aguas, habiendo sido detectada una
amplia variedad de estos compuestos y sus metabolitos en los sistemas acuáticos (Drewes et al.,
2001; Jones et al., 2005; Ternes, 2001). Por ejemplo, sustancias como la carbamazepina, atenolol,
metropopol, trimetoprim y diclofenaco, solo son eliminadas en las EDARs en porcentajes
inferiores al 10% (Ternes, 1998). Así mismo en publicaciones recientes, se indica que países como
España, Alemania, Canadá, Brasil, Grecia y Francia generan descargas al agua de
aproximadamente 500 toneladas/año en analgésicos, donde el ácido salicílico y el diclofenaco
alcanzan concentraciones de 0,22 a 3,02 µg l-1 (Heberer, 2002b) en los efluentes de EDARs. A
pesar de que las concentraciones detectadas en aguas superficiales y en aguas subterráneas se
sitúan normalmente en niveles traza, lo que ha despertado una mayor preocupación ha sido el
hallazgo de algunos de ellos (como el ibuprofeno, el diclofenaco, la carbamazepina o el ácido
clofíbrico) en aguas potables (Bedner and MacCrehan, 2005; Ternes et al., 2002).
15
CAPÍTULO 2
Además de que los tratamientos de aguas residuales convencionales no hayan sido

diseñados para la eliminación de fármacos, el hecho de que se trate de sustancias complejas de
muy diversa naturaleza, con grandes diferencias en su estructura y comportamiento físico-
químico, dificulta aún más su eliminación. Su persistencia en el medio ambiente puede ser mayor
de un año para fármacos como la eritromicina, ciclofosfamida, naxopreno, sulfametoxazol, y de
varios años para otros, como el ácido clofíbrico, que puede acumularse alcanzando niveles
biológicamente activos (Coimbra et al., 2015). Tras su administración, las moléculas son
absorbidas, distribuidas y, además, sujetas a reacciones metabólicas donde la estructura química
de la molécula activa puede ser modificada.
Los fármacos que se han detectado en el ambiente acuático, ya sea directamente o sus
metabolitos, incluyen los siguientes grupos terapéuticos (Hernando et al., 2006):
• Antiinflamatorios y analgésicos. Dentro de este grupo, los compuestos más

empleados son el paracetamol, ácido acetilsalicílico, ibuprofeno y diclofenaco.
• Antidepresivos. Los más frecuentes son las benzodiacepinas.
• Antiepilépticos. El más común es la carbamazepina.
• Antilipemiantes. Se aplican fundamentalmente, para bajar los niveles de colesterol
en sangre en personas con arterioesclerosis. Los fármacos más frecuentes son los
fibratos.
• β-bloqueadores. Utilizados principalmente para el tratamiento de los transtornos
del ritmo cardíaco. Los más utilizados son el atenolol, propanolol y metopropol.
• Antiulcerosos y antihistamínicos. Comúnmente, se emplean la ranitidina y
famotidina.
• Antibióticos. Entre los más importantes se encuentran las tetraciclinas, macrólidos,
β-lactámicos, penicilinas, quinolonas, sulfonamidas, fluoroquinolonas, cloranfenicol
y derivados imidazólicos.
2.4. EFECTOS Y RIESGOS DE LA PRESENCIA DE FÁRMACOS EN EL MEDIO AMBIENTE
Los fármacos, una vez liberados al medio ambiente, pueden tener efectos adversos aún
desconocidos sobre organismos vivos. Estos compuestos han sido desarrollados para actuar sobre
mecanismos metabólicos y moleculares en personas y animales. Al introducirse en el medio
ambiente, pueden actuar a través de los mismos mecanismos en animales que tengan órganos,
tejidos, células o biomoléculas similares. Sin embargo, en organismos inferiores los modos de
acción pueden sucederse de muchas otras formas. Además, algunos de estos fármacos tienen la
16
Antecedentes
capacidad de acumularse en los tejidos animales, siendo ejemplo de ello las concentraciones de
gemfibrozil, diclofenaco e ibuprofeno encontradas en el hígado, riñones y plasma de diversos
peces (Brown et al., 2007; Mimeault et al., 2005; Schwaiger et al., 2004).
En la actualidad, los efectos de la presencia de fármacos en el medio ambiente no son bien

conocidos. Las concentraciones detectadas en aguas naturales, no suelen presentar toxicidad
aguda sobre los organismos acuáticos (Kyungho et al., 2008). Sin embargo, el hecho de que
puedan sufrir una exposición prolongada a estos compuestos, despierta un gran interés sobre la
toxicidad crónica. Además, este riesgo se ve incrementado ya que, normalmente, estos
contaminantes se encuentran mezclados en el medio, provocando efectos combinados o
sinérgicos (Quinn et al., 2009). El uso de bioensayos permite evaluar la calidad de las aguas de
cara a la problemática ambiental asociada a los contaminantes emergentes en general y a los
fármacos en particular, ofreciendo información sobre biodisponibilidad y efectos conjuntos de
muestras ambientales completas en la biota. El uso de embriones de pez se ha utilizado en
recientes investigaciones para analizar posibles efectos ecotoxicológicos de los contaminantes en
el medio ambiente acuático (Caminada et al., 2006; Coimbra et al., 2015; Ribeiro et al., 2015;
Scholz et al., 2008; Soares et al., 2009; Torres et al., 2016). El pez cebra (Danio rerio) es una de las
especies más empleadas en estudios de ecotoxicidad y desarrollo genético (Ribeiro et al., 2015;
Soares et al., 2009; Torres et al., 2016). Estos bioensayos permiten la exposición prolongada a
contaminantes, evaluando los efectos causados en las distintas etapas del desarrollo embrionario,
así como en la edad adulta, mediante observación directa o mediante el empleo de biomarcadores
de daño. Además, no solo permiten evaluar la disminución de la toxicidad en el efluente tratado
por reducción de la concentración del fármaco, sino también verificar si existen productos de
transformación tóxicos tras el tratamiento.
Si aún no son bien conocidos los efectos en el ambiente, aún más compleja resulta la
identificación de los posibles efectos adversos que los contaminantes emergentes y,
específicamente los fármacos, pueden causar en la salud humana a través de su consumo en el
agua potable. Como ya se mencionó anteriormente, los contaminantes emergentes no necesitan
ser persistentes para encontrarse en el medio ambiente y ocasionar efectos negativos, ya que sus
altas tasas de transformación se ven compensadas por el continuo consumo e introducción en el
medio ambiente (Petrovic et al., 2003). Para la mayoría de este tipo de compuestos no existe
información sobre su incidencia, contribución al riesgo y efectos ecotoxicológicos, de tal manera
que es difícil predecir posibles riesgos para la salud humana.
17
CAPÍTULO 2
2.5. SISTEMAS DE TRATAMIENTO EN LA ELIMINACIÓN DE CONTAMINANTES

EMERGENTES DEL AGUA
Las plantas de tratamiento de aguas residuales han sido diseñadas para la eliminación de
un amplio rango de sustancias, como materia orgánica, nutrientes, patógenos y partículas en
suspensión. Sin embargo, tras la detección de nuevos contaminantes en las aguas y la entrada en
vigor de la Directiva 2008/105/CE, por la que se fijan las Normas de Calidad Ambiental para
algunos contaminantes emergentes clasificados como prioritarios, surgió la necesidad de valorar
la eficiencia de eliminación de estas sustancias mediante los sistemas de tratamiento
convencionales de aguas residuales.
Los contaminantes emergentes poseen propiedades químicas muy diversas, que hacen que
su grado de eliminación por los tratamientos convencionales pueda ser muy distinto, persistiendo
muchos de ellos inalterados en los efluentes de las plantas depuradoras. Por esta razón, es
importante identificar y evaluar tecnologías de tratamiento de aguas, que permitan una
incorporación segura de las aguas tratadas al medio acuático y eviten efectos adversos sobre el
medio ambiente y la salud humana.
2.5.1. Sistemas convencionales
Los sistemas de tratamiento de aguas residuales convencionales constan de tratamientos

físicos, químicos o biológicos según su modo de actuación; o de pre-tratamientos, tratamientos
primarios, secundarios, terciarios, etc. según el orden de actuación sobre el agua residual.
Los pre-tratamientos constan de operaciones mecánicas y físicas para la separación de

aquellas materias que por su naturaleza o por su tamaño puedan ocasionar problemas en etapas
posteriores (obstrucción de tuberías y bombas, depósitos de arena, rotura de equipos, etc.) Estas
operaciones constan habitualmente de una separación de grandes sólidos, desbaste, tamizado,
desarenado y desaceitado-desengrasado, mediante los cuales tan solo una pequeña fracción de
algunos contaminantes emergentes será eliminada, como son los compuestos liposolubles o los
que queden adsorbidos en la fracción sólida (Carballa et al., 2004).
Los tratamientos primarios tienen por objeto eliminar sólidos en suspensión y coloides. Los
sólidos en suspensión se podrán separar según su naturaleza por gravedad, flotación o filtración.
Sin embargo, debido a la naturaleza de las partículas coloidales, serán necesarios procesos de
coagulación-floculación para romper su estabilidad en el agua, y la posterior separación de los
agregados por decantación o flotación. La eliminación de contaminantes emergentes que pueda
ocurrir en esta etapa será de aquellos contaminantes adsorbidos sobre los lodos de la decantación
18
Antecedentes
primaria (Ternes et al., 2004). Carballa et al. (2004) alcanzaron una eliminación eficiente de
fragancias como galaxolide y tonalide durante el tratamiento primario (40%). El disruptor
endocrino nonilfenol monoetoxilado tan solo fue eliminado en un 13%, y el bisfenol A en un 43%
(Stasinakis et al., 2013). En el caso de fármacos y hormonas, la eliminación durante el tratamiento
primario solo alcanzó el 28% para el diclofenaco y el estriol, y no se observó ninguna reducción
para ibuprofeno, naproxeno, sulfametoxazol y estrona (Carballa et al., 2004). Por ello ha sido
afirmado que los tratamientos primarios no resultan efectivos en la eliminación de la mayoría de
los contaminantes emergentes (Carballa et al., 2005).
Los tratamientos secundarios consisten en diversos tratamientos biológicos que tendrán

por objeto degradar la materia orgánica biodegradable. Entre ellos se encuentran diferentes
sistemas como los lodos activos, los lechos bacterianos o los contactores biológicos rotativos
(biodiscos y biocilindros). Durante el tratamiento secundario los contaminantes emergentes
pueden sufrir diversos procesos y su eliminación generalmente se refiere a la pérdida del
compuesto parental debido a transformaciones químicas o físicas, biodegradación o adsorción
(Jelic et al., 2010). La biodegradación/biotransformación y la adsorción son los principales
mecanismos de eliminación en el tratamiento secundario, mientras que la volatilización ocupa
una menor fracción (Verlicchi et al., 2012). Salgado et al. (2012) observaron una baja
biodegradabilidad del diclofenaco (< 25%), mientras que el ibuprofeno y keroprofeno mostraron
una biodegradabilidad mucho mayor (> 75%). Por otra parte, los antibióticos no son fácilmente
biodegradables (Verlicchi et al., 2012).
Sin embargo, la configuración convencional de las EDARs no permite la eliminación

eficiente de estos nuevos contaminantes. En el caso particular de los fármacos, muchos de ellos no
son metabolizables por los microorganismos e incluso pueden llegar a inhibir su actividad. Por
otra parte, en los sistemas de cloración como tratamiento para la desinfección del agua, se ha
observado que en la eliminación del paracetamol se produce una reacción con cloro para formar
un gran número de subproductos, dos de los cuales han sido identificados como tóxicos (Bedner
and MacCrehan, 2005; Glassmeyer and Shoemaker, 2005). Por lo tanto, la mayor parte de estos
contaminantes no se eliminan eficientemente durante el tratamiento secundario, constituyendo la
principal vía de entrada de la contaminación de los medios acuáticos (Tabla 2.2).
19
CAPÍTULO 2
Tabla 2.2 Eficiencias medias de eliminación de fármacos detectados tras el tratamiento de las aguas residuales de
varias EDARs basadas en tratamientos convencionales (adaptación de Prados-Joya 2010).
Eficiencia de eliminación
Tipo de fármaco Sustancia detectada
media (%)
Ketoprofeno 29,5
Naproxeno 0
Analgésicos y
Ibuprofeno 48,5
antiinflamatorios
Diclofenaco 14,0
Paracetamol 79,4
Bezafibrato 56,5
Antilipemiantes Clofibrato 61,1
Gemfibrozil 22,6
Antiepilépticos Carbamazepina 2,4
Antiácidos Ranitidina 28,2
Azitromicina 36,8
Metronidazol 46,3
Antibióticos
Sulfametoxazol 33,9
Trimetoprima 75,3
Atenolol 1,3
β-bloqueadores Sotalol 9,7
Propanolol 42,1
2.5.2. Tratamientos avanzados
Los tratamientos avanzados buscan mejorar la calidad del agua mediante la eliminación de
la carga orgánica residual así como contaminantes no eliminados en el tratamiento secundario,
como son los nutrientes inorgánicos (nitrógeno y fósforo). A pesar de que el uso de estos
tratamientos está extendido en la eliminación de contaminantes específicos de los efluentes
industriales, su inclusión no resulta habitual como tratamientos terciarios en las EDARs. Estos
tratamientos pueden basarse en ósmosis inversa, electrodiálisis, destilación, coagulación,
adsorción mediante carbón activo, filtración, oxidación química, precipitación o nitrificación-
20
Antecedentes
desnitrificación. Además, los procesos como la desinfección con cloro, luz ultravioleta u ozono
permiten reducir el número de organismos vivos presentes en el agua.
Como se ha expuesto, los sistemas convencionales no resultan adecuados para la completa

eliminación de la gran variedad de contaminantes emergentes presentes en las aguas residuales.
Los procesos avanzados resultan una buena opción en el tratamiento de contaminantes
emergentes, sin embargo, la mayor parte de ellos presentan como desventaja su elevado coste y
dificultad de operación. Utilizándolos como tratamiento terciario en la configuración
convencional de las EDARs, permiten mejorar la biodegradabilidad de los contaminantes y
contribuir así a su eliminación. Sin embargo, el pH y la temperatura tienen una gran influencia en
su eliminación ya que influyen en la fisiología de los microorganismos y en la solubilidad de los
contaminantes en el agua (Cirja et al., 2008). Sin embargo, en la actualidad, no hay disponible
ningún tratamiento que asegure la completa eliminación de la mezcla de contaminantes
emergentes de las aguas residuales, por lo que se continúa investigando sobre diferentes
alternativas para lograrlo.
Coagulación-floculación
Los procesos de coagulación-floculación, a pesar de no ser un sistema nuevo, constituyen

una alternativa en su utilización como pre-tratamiento o como tratamiento terciario en los
sistemas convencionales de tratamiento de aguas residuales. En general, estos procesos no
resultan efectivos en la mayoría de los contaminantes emergentes, sin embargo, Suárez et al.
(2009) utilizaron sales de hierro y aluminio como pre-tratamiento sobre efluentes hospitalarios,
alcanzando eliminaciones elevadas para tonalide y galaxolide (< 79%), medias para diclofenco y
naproxeno (< 32%) y bajas para ibuprofeno, carbamazepina y sulfametoxazol (< 12%).
Matamoros y Salvadó (2013) aplicaron la coagulación-floculación a los efluentes del tratamiento
secundario obteniendo eficiencias medias (24-50%) para tonalide, celestolide, trilosan y
octilfenol; y bajas o inapreciables para ibuprofeno, kentoprofeno, carbamazepina, galaxolide,
dimetoxipropano (2-19%).
Sistemas de membranas
La eliminación de contaminantes mediante sistemas de membranas se logra a través de la

exclusión por tamaño, adsorción sobre la membrana y mediante cargas de repulsión (Luo et al.,
2014). La eliminación dependerá de diversos factores como las características de la membrana,
las condiciones de operación, las características propias del contaminante y de los fenómenos de
ensuciamiento de la membrana (Schäfer et al., 2011). Los sistemas de membrana comprenden
procesos a baja presión, como la microfiltración y ultrafiltración; y procesos a alta presión, como
21
CAPÍTULO 2
la nanofiltración y ósmosis inversa. Los sistemas de microfiltración y ultrafiltración no

presentaban eficiencias aceptables en la eliminación de contaminantes emergentes ya que el
tamaño de poro de la membrana no es capaz de retener estas moléculas (Luo et al., 2014). Sin
embargo, estos microcontaminantes pueden ser eliminados por adsorción a la membrana y por
interacciones con la materia orgánica del agua residual (Jermann et al., 2009).
Por otra parte, los sistemas de ósmosis inversa y nanofiltración son muy efectivos en la
separación física de contaminantes emergentes del agua (Bellona et al., 2008; Wei et al., 2015).
Ambos sistemas son utilizados en la industria para obtener agua de reutilización debido a sus
altas eficiencias, sin embargo sus membranas aún son permeables para algunos contaminantes
(Steinle-Darling et al., 2010). Así mismo, los procesos de electrodiálisis utilizados en las plantas de
desalinización, han comenzado a utilizarse en el ámbito de los contaminantes emergentes,
permitiendo que la corriente eléctrica que pasa a través de la membrana separe ciertos
compuestos del agua residual (Gabarrón et al., 2016; Guedes et al., 2016).
A pesar de que algunas técnicas de filtración a través de membrana a alta presión resultan
muy eficientes, implican altos costes operativos y de inmovilizado (Acero et al., 2010). Además,
presentan las desventajas de que las moléculas de bajo peso molecular continúan sin ser
retenidas y de que se genera una salmuera con una elevada concentración de fármacos. Para
solucionar el primero de estos problemas, se han realizado diversos estudios sobre la
combinación de procesos de filtración con membranas con procesos de coagulación o adsorción,
que permitan eliminaciones adicionales al aumentar el tamaño de aquellos compuestos con bajo
peso molecular que no serían retenidos por la membrana operando por sí sola (Acero et al., 2012;
Ang et al., 2016).
Reactores biológicos de membrana
Los reactores biológicos de membrana se consideran una mejora al tratamiento biológico

secundario, ya que combinan el proceso de los lodos activados con los sistemas de membranas.
Este sistema permite eliminar un amplio rango de microcontaminantes, ya que las membranas
son capaces de retener los lodos sobre los que los contaminantes han sido adheridos. En función
del tamaño de la membrana también podrá retener algunos de estos compuestos por sí mismas.
Además, al tener un mayor tiempo de retención del lodo, se logra una mayor degradación por
parte de los microorganismos (Spring et al., 2007). Por otro lado, presentan la ventaja de generar
una baja carga de lodos y mejor separación de la población microbiana. Sin embargo, presentan
una gran desventaja económica, lo que limita su aplicación.
22
Antecedentes
Trinh et al. (2012) alcanzaron una alta eliminación (<90%) mediante los sistemas de
membrana biológica para la mayoría de los contaminantes detectados. Sin embargo, algunos
fueron eliminados de forma incompleta (24-68%), entre los que se encontraban el diclofenaco,
carbamazepina, diazepan, gemfibrozil y omeprazol.
Adsorción mediante carbón activo
El carbón activo es comúnmente utilizado en el control del olor y sabor del agua potable.
Sin embargo, este método se ha evaluado como tratamiento terciario para la eliminación de
contaminantes emergentes por diversos autores (Campinas et al., 2016; Matamoros et al., 2009),
obteniendo eliminaciones superiores al 90% para diclofenaco, carbamazepina, propanolol,
triclosan, trimethoprim. La utilización del carbón activo en los procesos de adsorción presenta la
ventaja de no generar subproductos, evitando así el riesgo de formar productos de
transformación más contaminantes. Sin embargo, requiere grandes consumos de carbón activo,
que tiene un alto coste, y su eficacia disminuye en gran medida en presencia de materia orgánica
debido a la obstrucción de los poros por parte de esta última.
Los sistemas de adsorción con carbón activo combinados con ultrafiltración son tecnologías
prometedoras para el tratamiento de agua. En este sistema se suman los beneficios aportados por
la adsorción y la retención de partículas de alto peso molecular, por lo que permite la retención de
partículas de menor peso molecular que no serían eliminadas por la ultrafiltración por sí sola
(Acero et al., 2012).
Procesos de oxidación avanzada
Los procesos de oxidación se basan en la ruptura de las moléculas mediante el ataque a

grupos funcionales. La ozonización y los procesos de oxidación avanzada han demostrado ser
tecnologías eficientes por presentar altas tasas de degradación y por no ser selectivos, además de
presentar la ventaja de tener efectos desinfectantes (Hernández-Leal et al., 2011). Sin embargo,
presentan el inconveniente de suponer elevados costes de operación. Además, en el caso de los
contaminantes emergentes, y más aún en el de los fármacos, esto puede dar lugar a la
transformación de los contaminantes en otros compuestos de los que se desconocen sus efectos
sobre el medio ambiente y la salud humana (Evgenidou et al., 2015; Fatta-Kassinos et al., 2011).
Los procesos de oxidación avanzada implican la formación de radicales hidroxilo, altamente

reactivos, que actúan como iniciadores del proceso de oxidación. Entre estos procesos se
encuentra la fotocatálisis (empleando luz UV o solar en presencia de catalizadores
semiconductores) y los procesos Fenton (usan reacciones entre iones de hierro y peróxido de
23
CAPÍTULO 2
hidrógeno). Además, se ha investigado la eliminación de contaminantes emergentes mediante la

utilización de varios procesos de oxidación avanzada, como la combinación de ozono, radiación
UV y/o peróxido de hidrógeno, mejorando la degradación de fármacos polares y sus metabolitos
(Petrovic et al., 2003). Se ha comprobado que los procesos de fotocatálisis son capaces de eliminar
eficazmente diversos compuestos farmacéuticos, incluyendo los estrógenos (de la Cruz et al.,
2012; Deegan et al., 2011; Prieto-Rodríguez et al., 2012).
2.5.3. Tratamientos alternativos
En la Unión Europea ya se asumió la necesidad urgente de abordar el problema de la

contaminación por contaminantes emergentes y de desarrollar e implementar estrategias sostenibles y de
bajo coste para la remediación de aguas contaminadas por este tipo de compuestos (Barbosa et al.,
2016). Por ello, y en respuesta a los elevados consumos energéticos y costes operacionales de los
tratamientos avanzados, han surgido los llamados tratamientos alternativos, entendiendo como
tales aquellos que suponen bajos costes y son respetuosos con el medio ambiente.
Adsorbentes alternativos
Como ya ha sido referido, el carbón activado es un material altamente poroso debido a su

elevada área superficial, siendo el adsorbente más utilizado para el tratamiento de aguas.
El principal inconveniente de los carbones activados es su elevado precio. Aunque a nivel
industrial su utilización pueda ser factible, en el caso de las aguas residuales urbanas, debido a los
grandes volúmenes que es preciso tratar, los costes asociados a un tratamiento terciario de
adsorción utilizando carbón activado serían muy elevados. Por esta razón, en los últimos años,
han sido realizados numerosos estudios sobre la producción de adsorbentes alternativos y/o de
bajo coste a partir de bioresiduos y su utilización para la eliminación de fármacos de las aguas
(Bernardo et al., 2016; Coimbra et al., 2016; Ferreira et al., 2017; Tapia-Orozco et al., 2016).
Al igual que la adsorción sobre carbón activado, la eliminación de fármacos utilizando estos
adsorbentes alternativos tiene la ventaja de no llevar asociada la generación de productos de
transformación.
Fotodegradación con luz solar
La utilización de procesos de oxidación avanzada como tratamiento terciario en la

eliminación de fármacos presenta el inconveniente de elevados costes energéticos y
operacionales, además de poder formar productos de degradación tóxicos durante la oxidación
con ozono (Verlicchi et al., 2012). Como alternativa ha surgido la fotodegradación solar, que a
pesar de las variaciones estacionales, geográficas y climatológicas, presenta las ventajas de
24
Antecedentes
utilizar una fuente natural de oxidación, sostenible y de bajo coste (Kanakaraju et al., 2016). La luz
solar puede actuar directamente sobre los contaminantes o actuar de forma indirecta mediante la
formación de radicales (Mathon et al., 2016). Además de llevarse a cabo en los fotorreactores
solares, la fotodegradación solar puede tener lugar de forma simultánea a los tratamientos
biológicos. Norvill et al. (2017) estudiaron la eliminación de tetraciclinas de aguas residuales
mediante microalgas, resultando el principal mecanismo la fotodegradación. De la misma manera,
la fotodegradación solar tuvo un importante papel en la eliminación de contaminantes
emergentes en humedales artificiales (Ávila et al., 2015)
Tratamientos biológicos
Los sistemas biológicos de tratamiento de aguas residuales constituyen una alternativa

sostenible y económicamente rentable respecto a otros sistemas alternativos. Los sistemas de
tratamiento avanzados (procesos de oxidación avanzada, tecnología de membranas, etc.) suponen
un elevado consumo energético, elevados costes de construcción y mantenimiento y un efluente
de menor calidad ecológica que el obtenido mediante tratamientos biológicos (García-Rodríguez
et al., 2014).
Los sistemas de tratamiento de aguas residuales biológicos simulan la habilidad natural de

los ecosistemas para atenuar la contaminación del agua mediante procesos físicos
(adsorción/absorción, fotodegradación, volatilización y sedimentación), químicos (degradación e
hidrólisis) y biológicos (biodegradación y fitorremediación) (García-Rodríguez et al., 2014). Estos
mecanismos de eliminación se verán afectados por diversos factores, como la actividad de la
planta de tratamiento, la composición del agua residual, la acumulación de materia orgánica y los
cambios ambientales (García-Rodríguez et al., 2014).
Entre los tratamientos biológicos alternativos más importantes se encuentran los

humedales artificiales, las lagunas de estabilización y las lagunas de alta carga.
Los humedales artificiales se basan en estanques, lechos o zanjas de poca profundidad, que
contienen vegetación emergente propia de humedales que va a eliminar la contaminación por
mecanismos de biodegradación, fitorremediación, fotodegradación y adsorción a la matriz sólida.
En los últimos años han surgido numeras investigaciones sobre la eliminación de contaminantes
emergentes orgánicos mediante este sistema, variando en gran medida las eficiencias obtenidas
en función de factores como las dimensiones, tipo de vegetación, tiempo de retención hidráulica y
tipo de matriz sólida. Así las eficiencias detectadas por diversos autores varían entre 21-99% en
diclofenaco, 52-99% ibuprofeno, > 94% ácido salicílico, > 95% paracetamol, 65-99% bisfenol A,
< 28% carbamazepina, 45-99% naproxeno, 68% estrona, 84% β-estradiol y 75% 17-α-
25
CAPÍTULO 2
etinilestradiol (Ávila et al., 2013, 2010; Matamoros et al., 2009; Song et al., 2009; Zhang et al.,
2012a, 2012b).
Las lagunas de estabilización constituyen un sistema completo de depuración de aguas

residuales, constando de un estrato superior formado por algas y bacterias aerobias, uno inferior
constituido por bacterias anaerobias, y uno intermedio con la presencia de bacterias facultativas.
Los mecanismos de eliminación comprenden la fotodegradación, biodegradación y la adsorción
sobre la materia orgánica. Hijosa-Valsero et al. (2010) observaron una eliminación entre el 70 y
90% de fármacos y productos de cuidado personal (ibuprofeno, diclofenaco, ácido salicílico,
cafeína y metil dihidrojasmonato). Li et al. (2013) detectaron una mayor eliminación de algunos
de los compuestos evaluados (naproxeno, ibuprofeno, cafeína, triclosan, gemfibrocil,
sulfametoxazol) durante la estación cálida en este sistema de lagunajes, alcanzando eficiencias
entre 88-100%.
La biorremediación del agua mediante el cultivo de microalgas como alternativa a los

tratamientos convencionales, fue propuesta por primera vez por Oswald en la década de los 60
(Oswald et al., 1957). Desde ese momento, surgieron numerosas líneas de investigación en este
campo, entre las que destacan el aprovechamiento de la biomasa para la producción de
biocombustibles (Brennan and Owende, 2010; Christenson and Sims, 2011; Demirbas, 2011;
Maity et al., 2014), la eliminación de nutrientes (Arbib et al., 2013; Cabanelas et al., 2013; Gómez
et al., 2015; Morales-Amaral et al., 2015b; Posadas et al., 2015a), la eliminación de metales
pesados (Chen et al., 2012; Das et al., 2015; Fawzy and Issa, 2016; Puyen et al., 2012; Tao et al.,
2013; Vilar et al., 2009), la optimización de las condiciones de cultivo y diseño de biorreactores
(Chang et al., 2016; Imaizumi et al., 2016; Pfaffinger et al., 2016; Zheng et al., 2016); hasta su
utilización en la eliminación de contaminantes emergentes (Barceló and Petrovic, 2006; Carballa
et al., 2005; de Godos et al., 2012; Hom-Díaz et al., 2015; Matamoros et al., 2016).
2.6. UTILIZACIÓN DE LAS MICROALGAS EN EL TRATAMIENTO DE AGUAS RESIDUALES
La posibilidad de emplear microalgas para el tratamiento de aguas residuales se remonta a

los años 60, sin embargo a día de hoy, aún no se ha podido implantar como un sistema a escala
industrial ya que es necesario solventar ciertas limitaciones que impiden alcanzar la eficiencia de
los sistemas de tratamiento convencionales. Es por ello que, actualmente, no es posible plantear el
tratamiento con microalgas como un sistema de tratamiento por sí solo, sino como una etapa
dentro del sistema de tratamiento de una EDAR, bien como tratamiento secundario o bien como
26
Antecedentes
terciario, permitiendo cumplir los límites de vertido de nitrógeno y fósforo, así como la
eliminación de contaminantes emergentes.
El hecho de que las microalgas puedan ser cultivadas en agua residual se basa en la
similitud de la composición del agua residual con los nutrientes necesarios para el crecimiento de
las microalgas, siendo éstos principalmente carbono, nitrógeno y fósforo. Además, se encuentran
componentes minoritarios, como hierro, manganeso, etc. que son necesarios para la producción
de microalgas (Morales-Amaral et al., 2015b).
Una de las ventajas que presenta la utilización de microalgas en el tratamiento del agua
frente a los sistemas convencionales es la reducción de los costes energéticos, ya que a través de
la fotosíntesis se incrementa el oxígeno disuelto del agua, permitiendo reducir la aireación que
supone un 45-75% del coste total del proceso (Rosso et al., 2008). Además, permite rentabilizar el
proceso al obtener una biomasa de alto valor y destinarla a la producción de biofertilizantes,
producción de biogás y otros productos de biorrefinería. Por otra parte, las microalgas ayudan a
mitigar la emisión de gases de efecto invernadero que se producen en los tratamientos de aguas
residuales convencionales (dióxido de carbono, metano, óxido nitroso) (Acién et al., 2016).
Finalmente, como el más importante dentro del planteamiento en que se enmarca la presente
tesis, ofrece la ventaja de una eliminación efectiva de nutrientes inorgánicos (Acién et al., 2016;
Morales-Amaral et al., 2015b; Mujtaba et al., 2015), metales pesados (Das et al., 2015; Puyen et al.,
2012; Vilar et al., 2009) y ciertos contaminantes emergentes (Carballa et al., 2005; Hom-Díaz et
al., 2015; Matamoros et al., 2016), produciendo un efluente de alta calidad.
Aunque los cultivos de microalgas están formados por complejas poblaciones,

generalmente están dominados por una única especie que supone el 90% de la población de
microalgas (Acién et al., 2016). Las microalgas más empleadas en el tratamiento de algas
residuales, y que además se encuentran de forma natural, son las pertenecientes al género
Chlorella y Scenedesmus, debido a sus altas tasas de crecimiento y a su tolerancia a las condiciones
exteriores (Gómez et al., 2013). Además se han situado como uno de los géneros más tolerantes a
la contaminación en los sistemas de tratamiento de aguas residuales (Palmer, 1969).
27
CAPÍTULO 2
2.6.1. Implementación en los sistemas de tratamiento de aguas residuales

convencionales
Aunque la composición del agua residual varía en función de su localización y de las

actividades predominantes en la zona (actividad urbana, agrícola, ganadera, industrial, etc.),
contiene en todo caso materia orgánica, nutrientes, microorganismos, toxinas, metales pesados y
contaminantes emergentes (Morales-Amaral et al., 2015b). Además, aunque las aguas residuales
contienen generalmente los componentes mayoritarios para el cultivo de las microalgas (carbono,
nitrógeno, fósforo), debido a la variación en su composición y a las variaciones estacionales, es
necesario estudiar cada caso de forma individual (Christenson and Sims, 2011; Rawat et al.,
2011). Los micronutrientes como el silicio, calcio, magnesio, potasio, hierro, manganeso, azufre,
zinc, cobre y cobalto, no suelen ser un factor limitante para su cultivo, en el caso de las aguas
residuales.
El carbono a su entrada en la EDAR, estará en forma de materia orgánica, el nitrógeno

aparecerá principalmente en forma de urea, amonio, y nitrógeno orgánico; mientras que el fósforo
estará presente como fosfato y fósforo orgánico.
La composición de la biomasa de las microalgas se corresponde con un 40-60% de carbono,

4-12% nitrógeno y 0,5-2% fósforo (Cabanelas et al., 2013; Posadas et al., 2014);
correspondiéndose con una relación C/N/P que puede variar entre 100/20/3 a 100/10/1 en
función de la especie y las condiciones de cultivo. En función de la etapa del tratamiento de agua
residual convencional en que queramos implementar un sistema de tratamiento con microalgas,
se deberá tener en cuenta la limitación o exceso existente de alguno de los nutrientes que
impedirá la eficiente eliminación simultánea. La relación C/N/P del efluente del tratamiento
primario, del secundario, así como del residuo líquido procedente del filtrado de la digestión
anaerobia (Tabla 2.3), demuestran que en todos ellos hay una deficiencia de carbono que no
permitiría la completa asimilación de nitrógeno y fósforo (Acién et al., 2016). Esta limitación se
puede solventar mediante la adición de bicarbonato o la inyección de CO2 a demanda de gases de
combustión, siendo necesario además para el control del pH.
28
Antecedentes
Tabla 2.3 Composición del agua residual en carbono, nitrógeno y fósforo total en diferentes etapas de una EDAR
convencional (adaptación de Acién et al. 2016).
Tratamiento Efluente de la digestión

Concentración (mg l-1) Tratamiento secundario
primario anaerobia de lodos
Carbono 296 82 247
Nitrógeno 65 20 511
Fósforo 11 10 12
C/N/P 100/21/4 100/25/12 100/207/5
El efluente procedente del tratamiento primario contiene las proporciones adecuadas para
el cultivo de microalgas sin que sea necesario realizar modificaciones. Se han llevado a cabo
numerosos estudios en el que el consorcio microalgas-bacterias se ha propuesto como
tratamiento secundario (AlMomani and Örmeci, 2016; Lv et al., 2016; Posadas et al., 2015a).
Además, las microalgas pueden implementarse como tratamiento terciario en aquellos casos en
los que se necesite reducir aún más los niveles de nitrógeno y fósforo del tratamiento secundario
para cumplir con las estrictas regulaciones, además de eliminar contaminantes emergentes y
patógenos (AlMomani and Örmeci, 2016; Gao et al., 2016; Gómez et al., 2013; Matamoros et al.,
2015). En este caso, debido a la baja concentración de nutrientes, sería necesario usar membranas
que permitieran retener las células y aumentar el flujo de agua residual (Acién et al., 2016).
Finalmente, numerosos estudios se han llevado a cabo para el tratamiento del efluente
procedente de la digestión anaerobia de la línea de lodos (AlMomani and Örmeci, 2016; Ledda et
al., 2015; Morales-Amaral et al., 2015a; Posadas et al., 2015a; Sepúlveda et al., 2015), ya que
contendrá un alto contenido en nutrientes y contaminantes. Sin embargo, estudios recientes
(Morales-Amaral et al., 2015b) demuestran que es necesaria una dilución previa (40-50%) antes
del cultivo de microalgas ya que el exceso de amonio (> 100 mg l-1) puede inhibir su crecimiento
(Collos and Harrison, 2014). A pesar de este efluente tan solo supone el 2% del caudal total de
agua residual, su recirculación a la cabecera de la EDAR, supone un incremento sustancial de los
costes y del consumo de energía.
En todas estas etapas de tratamiento, se debe tener en cuenta la importante sinergia entre
microalgas y bacterias. Las bacterias serán las responsables de oxidar la materia orgánica para
dejar disponibles los compuestos inorgánicos a las microalgas (dióxido de carbono, amonio,
fosfatos), mientras que las microalgas a través de la fotosíntesis consumirán estos compuestos
inorgánicos para producir biomasa e incrementarán el oxígeno disuelto del agua que utilizarán las
bacterias en la oxidación de la materia orgánica (Figura 2.3). Sin embargo, este equilibrio no
29
CAPÍTULO 2
resulta tan sencillo ya que las microalgas pueden además tener un comportamiento mixotrófico,
asimilando ciertas moléculas orgánicas. Además, los metabolismos de las bacterias aerobias
heterótrofas responsables de la degradación de la materia orgánica, así como las bacterias
aerobias autótrofas nitrificantes y las bacterias anóxicas heterótrofas desnitrificantes, tendrán
lugar de forma simultánea. Debido a estas interacciones, la relación microalgas-bacterias se trata,
en realidad de un complejo sistema cuyas interacciones continúan siendo estudiadas para poder
construir un modelo (Broekhuizen et al., 2012; Karya et al., 2013).
Figura 2.3 Esquema simplificado de la relación microalgas-bacterias en el tratamiento de aguas residuales

(fuente: elaboración propia).
A pesar de las numerosas ventajas que ofrece el cultivo de microalgas en el tratamiento de

aguas residuales y las altas eficiencias obtenidas en la eliminación de nutrientes (Cabanelas et al.,
2013; Gómez et al., 2013; Morales-Amaral et al., 2015b) y contaminantes emergentes (de Godos et
al., 2012; de Wilt et al., 2016; Matamoros et al., 2016), es innegable admitir que existen diversas
limitaciones que deben ser mejoradas para poder sustituir los sistemas de tratamiento
convencionales. Sin embargo, constituyen una alternativa viable como tratamiento
complementario. Estos factores limitantes comprenden la susceptibilidad de las microalgas a la
variación de la composición del agua residual (principalmente metales pesados y contaminantes
emergentes) y a las variaciones en las condiciones ambientales. Además, es necesario reducir el
tiempo de retención hidráulica desde 7-11 días a los 0,3 días utilizado en los procesos
convencionales y reducir el consumo de energía por debajo de 0,5 kWh/m3 (Acién et al., 2016).
30
Antecedentes
2.6.2 Mecanismos de eliminación de nitrógeno y fósforo
Las microalgas pueden asimilar el nitrógeno en forma de NO3-, NO2- o NH4+, siendo todas las
formas reducidas a amonio antes de ser incorporados en aminoácidos (Figura 2.4). Por lo tanto,
las formas de nitrato y nitrito serán consumidas tras el agotamiento del amonio, teniendo
preferencia por esta última forma de nitrógeno ya que requiere un menor consumo de energía
(Cai et al., 2013). Esto representa una ventaja en el cultivo en aguas residuales ya que la mayor
parte del nitrógeno total está en forma de amonio. Sin embargo, a veces puede ocurrir que el
oxígeno disuelto (OD ≥ 2 mg O2 l-1) provoque una rápida oxidación del NH4+ a formas de NO3-,
siendo superior que la asimilación de NH4+ por las microalgas o la volatilización de NH3; de tal
forma que finalmente la asimilación del nitrógeno sea en forma de NO3-, suponiendo mayores
requerimientos de energía y una disminución de la eficiencia de eliminación (Cai et al., 2013;
Posadas et al., 2015b). Además, como mecanismo abiótico durante el cultivo de microalgas, la
eliminación de nitrógeno también puede ocurrir por la volatilización de NH3 en condiciones de
elevado pH y alta concentración de NH4+. Sin embargo, bajo estas condiciones el crecimiento de
las microalgas puede quedar inhibido (Acién et al., 2016).
El fósforo en forma de PO43- se eliminará por asimilación a la biomasa, o de forma abiótica

por precipitación en condiciones de elevado pH (> 9) (Cai et al., 2013; Heubeck et al., 2007). Sin
embargo, debido al control de las condiciones de cultivo, este último mecanismo no tiene lugar
cuando se opera bajo condiciones de pH óptimo. Teniendo en cuenta que la composición de
fósforo de la biomasa varía de 0,5 a 2%, éste podrá ser acumulado como polifosfato para usarlo
posteriormente como una reserva de fósforo para el crecimiento de la biomasa cuando se
encuentre de forma limitada en el medio o como fuente de energía (Brown and Shilton, 2014;
Powell et al., 2008).
31
CAPÍTULO 2
Figura 2.4 Degradación bacteriana del nitrógeno y formas de asimilación en las microalgas
(fuente: elaboración propia).
2.6.3. Mecanismos en la eliminación de contaminantes emergentes
Los principales mecanismos que tienen lugar en la eliminación de contaminantes

emergentes mediante microalgas son la adsorción/absorción y la biodegradación. Sin embargo, el
conocimiento en este campo aún es escaso ya que depende de múltiples factores.
Los procesos de absorción/adsorción de los contaminantes emergentes dependen en gran

medida de la estructura del compuesto considerado (hidrofobicidad y grupos funcionales
disponibles), de la especie de microalga que actuará como adsorbente/absorbente y de las
condiciones ambientales (pH y temperatura) (Norvill et al., 2016). Además, la adsorción parece
ser un paso previo en la biodegradación de algunos contaminantes (Shi et al., 2010; Urase and
Kikuta, 2005). Wilt et al. (2016) observaron una eliminación debida a procesos de adsorción
mediante el cultivo de Chlorella sorokiniana < 20% para los fármacos diclofenaco, ibuprofeno,
paracetamol, metopropol, carbamazepina y trimetopin. En el caso de los estrógenos, diversos
autores han obtenido eliminaciones < 10% tanto por adsorción a biomasa de microalgas muerta
(Hom-Díaz et al., 2015; Zhang et al., 2014) como viva (Shi et al., 2010). Norvill et al. (2017)
determinaron una degradación del antibiótico tetraciclina del 93-99%, siendo el principal
mecanismo la fotodegradación y tan solo un 6% de la eliminación total fue atribuible a la
adsorción del fármaco a la biomasa de microalgas y bacterias.
32
Antecedentes
La biodegradación de contaminantes orgánicos emergentes por medio de microalgas puede

tener lugar de forma directa mediante metabolismo heterótrofo (Neilson and Lewin, 1974;
Subashchandrabose et al., 2013, 2011) o mediante la acción de enzimas extracelulares (Wurster
et al., 2003). Además, de una forma indirecta estos compuestos pueden ser degradados por el
incremento del oxígeno disuelto en el agua, fotosintéticamente mediante cambios de pH, o en su
caso por la relación simbiótica establecida con las bacterias (Muñoz and Guieysse, 2006; Norvill et
al., 2016). Existen pocos estudios publicados sobre la eliminación de contaminantes emergentes
por microalgas, sin embargo, la mayoría de ellos apuntan hacia una significativa eliminación
mediante biodegradación. Shi et al. (2010) observaron una biodegradación > 52% de estrona,
17β-estradiol y 17α-etinilestradiol. Hom-Díaz et al. (2015) comprobaron que de una eliminación
entre el 60-100% de 17β-estradiol y 17α-etinilestradiol, tan solo un 20-54% era atribuible a la
biodegradación. Además, Matamoros et al. (2015) atribuyeron la eliminación de 26
contaminantes emergentes a la biodegradación y fotodegradación, aunque en función del
compuesto, las eficiencias variaron entre insignificantes a > 90%.
2.6.4. Separación de la biomasa
Desde el punto de vista del cumplimiento de las regulaciones de calidad del agua, es
necesario separar la biomasa del efluente tratado antes de su vertido. En el caso de los
tratamientos mediante microalgas, esta separación representa una oportunidad para rentabilizar
el proceso del tratamiento del agua residual debido a la posterior utilización de la biomasa de
microalgas.
La sedimentación natural de las microalgas presenta valores inferiores a 10-6 m s-1

(Granados et al., 2012). Esto es debido al pequeño tamaño celular (< 30 µm), su baja
concentración en el cultivo (0,5-2,0 g l-1) y las fuerzas electrostáticas de repulsión entre las células
que hace que se mantengan en suspensión (Grima et al., 2003).
Diversas técnicas pueden ser aplicadas para la separación o cosechado de las microalgas,
tales como centrifugación, filtración, sedimentación, flotación, coagulación-floculación y métodos
electrolíticos (Granados et al., 2012). Sin embargo, debido a los grandes volúmenes de agua
procesados en una EDAR, es necesario emplear un sistema que sea económicamente viable.
La recuperación de la biomasa mediante coagulación-floculación combinada con

decantación/flotación junto con un posterior desecado de la biomasa mediante centrifugación o
sedimentación ha sido presentado como el sistema más adecuado para su aplicación en el
tratamiento de aguas residuales (Christenson and Sims, 2011; Schenk et al., 2008). Los floculantes
33
CAPÍTULO 2
utilizados en los procesos convencionales del tratamiento del agua, como son sales de aluminio,
hierro o polielectrolitos, han demostrado ser efectivos para este propósito (Granados et al., 2012),
pues permiten obtener un cosechado con una concentración de biomasa de 30-40 g l-1 y
eficiencias superiores al 95 % (Acién et al., 2016).
En el caso del tratamiento de aguas residuales mediante microalgas, se obtendrán grandes

cantidades de biomasa en función de la disponibilidad de nutrientes, pudiendo llegar a alcanzar
1 kg de biomasa por cada metro cúbico de agua residual procesada, siendo este valor 5 veces
superior al que se obtendría de los lodos en tratamientos convencionales (Acién et al., 2016).
Debido a la gran cantidad de biomasa producida, es necesaria una adecuada gestión para que
suponga un beneficio y no una limitación a la implantación de las microalgas en el tratamiento de
aguas residuales. Una alternativa es la digestión anaerobia de la biomasa para producir biogás
junto con los sistemas convencionales utilizados en la línea de lodos de una EDAR. Sin embargo,
son necesarios pre-tratamientos de la biomasa con el objetivo mejorar su biodegradabilidad
(Doğan-Subaşı and Demirer, 2016; Heaven et al., 2011; Passos and Ferrer, 2015). Otra de las
opciones pasa por producir biofertilizantes y bioestimulantes para uso agrícola, debido al alto
contenido en nitrógeno de la biomasa. Es necesario un tratamiento, como la hidrólisis enzimática,
para liberar los compuestos y estabilizar la biomasa para su uso comercial (Romero García et al.,
2012). Además de proporcionar nutrientes, la biomasa de microalgas es una fuente de
fitohormonas y estimulantes del crecimiento (García-González and Sommerfeld, 2016), por lo que
la biomasa tiene un alto valor agrícola. Actualmente se está investigando la utilización de este tipo
de biomasa como alimento para animales, productos químicos, bioplásticos o biocombustibles
(Hwang et al., 2016; Zeller et al., 2013).
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50

Capítulo 3

Objetivos

Objetivos

3.1. OBJETIVOS
La creciente preocupación social y científica derivada de la presencia de fármacos como

contaminantes emergentes en las aguas deja patente la necesidad de investigar en tratamientos
alternativos, sostenibles y de bajo coste. En este sentido, tal y cómo se ha mostrado en la
introducción general y en los antecedentes, la aplicación de las microalgas como sistemas de
biorremediación para en la eliminación de nutrientes de las aguas residuales ha sido ampliamente
estudiada. Sin embargo, la capacidad de las microalgas para eliminar fármacos del agua ha sido aún
poco investigada. A pesar de que estos tratamientos han sido considerados como prometedores, es
necesario investigar para continuar recabando información. En este contexto, el principal objetivo
de la presente tesis ha sido estudiar la aplicación de las microalgas como sistema de
biorremediación de aguas contaminadas por nutrientes y fármacos. Para alcanzar este objetivo
fueron planteados los siguientes objetivos específicos:
i. Determinar si el crecimiento de Chlorella sorokiniana, Chlorella vulgaris y Scenedesmus

obliquus se ve afectado por la presencia de los fármacos paracetamol, ácido salicílico y
diclofenaco.
ii. Evaluar la eficiencia de eliminación de nitratos y fosfatos mediante microalgas en

cultivo discontinuo y semicontinuo, estudiando además si dicha eficiencia se ve
influenciada por la presencia de fármacos.
iii. Determinar la eficiencia de eliminación de los fármacos paracetamol, ácido salicílico y

diclofenaco en cultivo discontinuo y semicontinuo mediante el cultivo de microalgas.
iv. Estudiar la aplicación de la microalga Chlorella sorokiniana como sistema de

tratamiento de fármacos en altas concentraciones relativas, en cultivo discontinuo y
semicontinuo.
v. Evaluar la separación de la biomasa de microalgas del agua tratada mediante

coagulación‐floculación, estudiando diferentes agentes floculantes en función de la
especie de microalga y modo de cultivo.
vi. Determinar la eficiencia de las microalgas en la disminución de la toxicidad del agua

tras la biorremediación de fármacos mediante el estudio de los efectos producidos en
el desarrollo embrionario de pez cebra (Danio rerio).
Los objetivos anteriores fueron abordados en las publicaciones que constituyen la tesis.
Específicamente, los objetivos i, ii, iii fueron contemplados en los Artículos I, III y IV; el objetivo iv
en el Artículo II; el objetivo v en el Artículo I y III; mientras que el Artículo V se centró en el objetivo
vi.
53

Capítulo 4
Artículo I
Nutrients and pharmaceuticals removal from wastewater by
culture and harvesting of Chlorella sorokiniana
C. Escapa, R.N. Coimbra, S. Paniagua, A.I. García, M. Otero

Bioresource Technology, 185 (2015): 276-284
Bioresource Technology 185 (2015) 276–284
Contents lists available at ScienceDirect
Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech
Nutrients and pharmaceuticals removal from wastewater by culture

and harvesting of Chlorella sorokiniana
C. Escapa ⇑, R.N. Coimbra, S. Paniagua, A.I. García, M. Otero
Department of Applied Chemistry and Physics, Institute of Environment, Natural Resources and Biodiversity (IMARENABIO), University of León, 24071 León, Spain
h i g h l i g h t s g r a p h i c a l a b s t r a c t
Removal of nitrates and phosphates

was close to 100% at the end of the
batch.
Phosphates removal was higher than
nitrates in the semicontinuous
culture.
Removal of salicylic acid and
paracetamol was around 70% at the
end of the batch.
Removal rates were 2.3 times higher
for salicylic acid than for paracetamol.
AlCl3 provided efficiencies above 95%
in 1 min with 200 mg g1 dose.
a r t i c l e i n f o a b s t r a c t
Article history: This work aimed to study both the removal of nutrients and pharmaceuticals, namely salicylic acid or
Received 22 January 2015 paracetamol, from water by the culture of Chlorella sorokiniana. The removal of nutrients was nearly com-
Received in revised form 26 February 2015 plete at the end of the batch culture; above 70% for nitrates and 89% for phosphates in the semicontinu-
Accepted 1 March 2015
ous culture. The pharmaceuticals removal kinetics were 2.3 times greater for the salicylic acid than
Available online 6 March 2015
paracetamol, reaching volumetric efficiencies above 93% for salicylic acid in the semicontinuous culture.
Finally, to separate the microalgae biomass from treated water, metal salts, synthetic polyelectrolytes
Keywords:
and a biopolymer were tested as coagulants–flocculants. The best flocculation results were achieved with
Microalgae
Emerging contaminants
AlCl3 (95.23% with 200 mg g1, 1 min incubation time). However, given that resulting flocs had different
Acetaminophen characteristics, flocculants must be chosen on the basis of the subsequent use of the biomass.
Salicylic acid Ó 2015 Elsevier Ltd. All rights reserved.
Coagulation–flocculation
1. Introduction at trace concentrations (lg l1, ng l1) in natural waters, their

potential adverse effects have increased scientific and social con-
The recent development of more sensitive analytical methods cern about the risks for human health and environment.
has allowed the detection and quantification of new pollutants in Owing to the continuous introduction in the environment and
natural waters, whose presence in the environment is not their cumulative effect, these emerging contaminants (ECs) have
necessarily new. Even though these compounds are often found been detected in natural waters, wastewaters and drinking water.
These ECs include pharmaceuticals, personal care products, surfac-
tants, industrial additives and various chemicals that, coming from
⇑ Corresponding author at: Department of Applied Chemistry and Physics, different origins, are discharged either into natural streams either
Institute of Environment, Natural Resources and Biodiversity (IMARENABIO),
into sewers (Bolong et al., 2009). In fact, their presence in natural
University of León, Avenida de Portugal 41, 24071 León, Spain. Tel.: +34 987293378.
E-mail addresses: carla.escapa@unileon.es (C. Escapa), ricado.decoimbra@
waters is mostly related to the inability of conventional sewage
unileon.es (R.N. Coimbra), spanb@unileon.es (S. Paniagua), ana.garcia@unileon.es treatment plants (STPs) to remove these ECs and their metabolites
(A.I. García), marta.otero@unileon.es (M. Otero).
http://dx.doi.org/10.1016/j.biortech.2015.03.004
0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
57
C. Escapa et al. / Bioresource Technology 185 (2015) 276–284 277
from wastewater. As a result, these ECs are discharged into the filtration, gravity sedimentation, flotation and electrolytic meth-
receiving streams and STPs constitute a relevant source of these ods) (Granados et al., 2012), flocculation coupled with flotation/
pollutants for the environment. sedimentation process and subsequent further dewatering by cen-
Of particular concern is the presence of pharmaceuticals on trifugation or sedimentation, seems to be the most promising
account of their accumulation in tissues, reproductive damage, alternative (Schenk et al., 2008).
inhibition of cell proliferation and behavioural changes in organ- Several coagulants–flocculants have been studied extensively
isms for which they were not intended (Escher et al., 2011). (Rakesh et al., 2014; Granados et al., 2012; Sirin et al., 2012;
Among pharmaceuticals, paracetamol is easily accumulated in Papazi et al., 2010). Metal salts are used in wastewater treatment
aquatic environment due to its relative high solubility and process due to their high efficiency. Polyelectrolytes, which are
hydrophilicity. Moreover, it has been confirmed that 58–68% of also used in wastewater treatments, attached cells by physical or
paracetamol and their metabolites are excreted from the body dur- chemical linking, are required at lower doses, are biodegradable
ing therapeutic use (Muir et al., 1997). On the other hand, salicylic and have no toxic effects (Granados et al., 2012). In the last years
acid removal from aqueous solutions has received a great deal of have been emerged biodegradable organic compounds as chitosan,
attention in recent years due to its high toxicity and accumulation starch, alginic acid or guar gum; which are biodegradable and have
in the environment (Combarros et al., 2014). Furthermore, it is a no toxic effects on the recovery biomass (Singh et al., 2000).
common derivate of phenol and, therefore, it is a typical pollutant Therefore, the aim of this study was to determine the biore-
in industrial wastewaters. mediation potential of Chlorella sorokiniana to remove paracetamol
Wastewater from the pharmaceutical industry are mainly trea- and salicylic acid from water and if the presence of these pharma-
ted by chemical oxidation methods, but the harsh reaction condi- ceuticals affected microalgal growth. Furthermore, the simultane-
tions, the generation of secondary pollutants and high ous elimination of nitrates and phosphates, was also assessed.
operational cost associated, may made of these treatments a not Finally, different coagulants–flocculants were used at different
a desirable option (Wu et al., 2012). On the contrary, biodegrada- dosages to test their efficiency in the subsequent microalgae bio-
tion treatments are considered as an environmentally friendly mass recovery.
and low-cost choice. Some microorganisms have developed spe-
cialized enzymatic systems and metabolic pathways to access
2. Methods
pharmaceuticals as carbon and energy source, and converting
them to easily metabolizable substrates (Wu et al., 2012).
2.1. Microorganism and culture conditions
Despite there are preliminary studies about biodegradation of
pharmaceuticals by microorganisms (Combarros et al., 2014;
The microalgae strain used in this study was C. sorokiniana CCAP
Zhang et al., 2013; Wu et al., 2012; De Gusseme et al., 2011), to
211/8 K (UTEX Culture Collection). Inoculum for the experiments
our best knowledge there is not information available about biode-
was cultivated in 250 ml Erlenmeyer flasks in the standard culture
gradation of pharmaceuticals by microalgae.
medium Mann and Myers (Mann and Myers, 1968). The growth
On the other hand, it should be kept in mind that one of the
conditions were maintained constant, under controlled tempera-
pressing goals in a wastewater treatment is the need to remove
ture (25 ± 1 °C), irradiance (175 lE m2 s1) and photoperiod
high concentrations of nitrogen and phosphorus, up to the limits
(12:12).
set by the European Commission Directive 98/15/EEC. It is well-
Experiments were carried out in bubbling column PBRs with
known that microalgae can play an important role in phytore-
spherical bases (40 mm diameter and 300 mm height with
mediation of wastewaters. Extensive research have been con-
300 ml capacity), keeping an operating volume of 250 ml. All reac-
ducted to determine the feasibility of cultivating microalgae in
tors were inoculated with the same volume of pre-cultured
wastewater and removing organic carbon and inorganic nutrients,
microalgae to ensure the same initial concentration, about 3.106 -
such as nitrogen and phosphorus from these polluted effluents.
cells ml1. Culture conditions were kept constant under controlled
Gómez et al. (2013) reported the feasibility of the biomass produc-
in temperature (25 ± 1 °C), irradiance (370 lE m2 s1) and pho-
tion of Muriellopsis sp. using secondary-treated wastewater cou-
toperiod (12:12), inside a vegetal culture chamber. Culture med-
pled with the depuration wastewater process. This system
ium was the same used for the inoculum growth. PBRs were
allowed a reduction in the biomass production costs by decreasing
illuminated by 8 fluorescent lamps (58 W, 2150 lumen, Philips,
freshwater and nutrients consumption, and provided simultane-
France) and aerated at a rate of 0.3 v/v/min with CO2 enriched at
ously a depuration process. Under these conditions, the culture
7% v/v, which was injected on demand to keep a constant pH
was nitrogen-limited which supposed a metabolism directed at
(pH = 7.5 ± 0.5) as controlled by a pH sensor. Before injection, the
carbohydrates and lipids accumulation. Domínguez Cabanelas
air was filtered through 0.2 lm sterile air-venting filter (Millex-
et al. (2013) researched about the use of several streams from sev-
FG50, Millipore).
eral stages of municipal wastewater as culture medium for
Chlorella vulgaris production. The bests results about biomass pro-
duction were obtained with the centrate, with removal rates of 9.8 2.2. Removal of pharmaceuticals and nutrients
(N) and 3.0 (P) mg l1 day1. Furthermore, the removal of nitrogen
and phosphorus from wastewater by microalgae culture has been 2.2.1. Experimental set-up
extensively studied and results indicate that this culture may be Reactors were operated in batch mode until the end of the
a promising bioremediation process (Arbib et al., 2013; Aslan and exponential growth, following by a semicontinuous mode until
Kapdan, 2006; de-Bashan et al., 2002). the culture parameters remained constant. The daily dilution rate
After wastewater treatment by microalgae, it is necessary to in this state was 30% of the culture volume, which was replaced
separate the biomass from the treated water. This separation also with fresh medium. Experiments were carried out simultaneously
allows biomass recovery for further potential use as feedstock for in all the reactors, under identical conditions.
biodiesel, bioactive molecules, pigments and nutraceuticals Two types of experiments were carried out: inoculated culture
(Gupta et al., 2013). The contribution of biomass recovery to the medium with 25 mg l1 paracetamol (PC) and inoculated culture
total cost of the biomass production has been estimated at 20– medium with 25 mg l1 salicylic acid (SaC). In each case, trials also
30% (Grima et al., 2003). Even though several techniques have been included a positive control with inoculated culture medium (C+)
applied in harvesting microalgae (centrifugation, flocculation, and the corresponding negative control with 25 mg l1 PC (CP-)
58
278 C. Escapa et al. / Bioresource Technology 185 (2015) 276–284
or 25 mg l1 SaC (CSa-). Paracetamol (C8H9NO2) was supplied by 2.3. Coagulation–flocculation

Roic Pharma and salicylic acid (C7H6O3) by Panreac. All assays were
performance in triplicate. 2.3.1. Experimental set-up
Throughout the experiments, the culture growth was daily Culture remained in batch mode until the exponential growth
monitored by biomass concentration and cell density. The concen- finished, followed by semicontinuous mode with a 30% dilution
tration of nitrate and phosphate was determined at the beginning rate of culture volume.
and the end of the batch culture and daily during the semicontinu- Metal salts, synthetic polyelectrolytes and a biopolymer were
ous culture. Moreover, in order to assess the removal of pharma- tested to determine the minimum dosage that ensures recovery
ceuticals, the concentration of paracetamol and salicylic acid was efficiencies of biomass higher than 95%. Iron chloride (FeCl3) and
daily monitored. aluminium chloride (AlCl3) were used as metal salts, supplied by
Panreac. Synthetic polyelectrolytes were supplied by Chemipol,
2.2.2. Analytical methods sold as chemifloc CH-35 (strong cationic polymers) and chemifloc
Biomass concentration (Cb) was determined by optical density CH-15 (medium cationic polymers), both with high molecular
at 680 nm (OD680) by spectrophotometric (UV/visible spectropho- weight and being used in industrial and urban wastewater treat-
tometer BECKMAN DU640) and verified by dry weight. ment processes. Chitosan was supplied by Acofarma as biopoly-
Preliminary studies were conducted to determinate the relation- mer. Doses of each coagulant were expressed as mg of coagulant
ship between dry weight and OD680 as shown in Eq. (1): per g of microalgae biomass.
Flocculation assays were performed in test-tubes using 25 ml
OD680 ¼ 5:1834 C b þ 0:0128; R2 ¼ 0:9983 ð1Þ harvest volume. Flocculants were previously dissolved in water;
metal salts and chitosan were dissolved at 10 mg ml1, and syn-
Dry weight measurements were performed by filtering 10 ml of
thetic polyelectrolytes at 2 mg ml1. To ensure complete solubility
culture through a 0.45 lm Whatman filter and washing with 20 ml
of the coagulant and to favour more collisions between colloids
HCl (0.5 M) to dissolve precipitated salts. Then, the filtrate was
and their possibility of aggregation, each dose was added under
dried in an oven at 80 °C for 24 h. Additionally, the growth of the
intense stirring at 150 rpm during 2 min. Following this, the stir-
culture was measured as cell density (Nc) by cell counting with a
ring was reduced (80 rpm, 5 min) to favour the formation of flocs
Neubauer chamber.
and avoid their mechanical breakage. After that, the suspension
Nitrates (N-NO3) were measured by the ultraviolet spec-
was placed in the test-tube, in which the flocculation efficiency
trophotometric screening method, according to Standard
was measured after 0, 1, 5, 10, 15, 30 and 60 min. Samples of
Methods 4500-NO 3 B (APHA, 2008); and phosphates were mea-
1 ml were taken from the clarified zone and the biomass concen-
sured by the ascorbic acid method, according to the Standard
tration was measured by absorbance at 680 nm.
Methods 4500-P E (APHA, 2008).
A first series of experiments was performed to study the effi-
A Waters HPLC 600 E equipped with a 2996 Photodiode Array
ciency of five doses of each coagulant with the biomass harvested
Detector was used for the quantification of the target pharmaceu-
at the end of the batch culture. Then, on the basis of the results
ticals. The wavelengths of detection were 246 and 236 nm for
from this first stage, a second series of trials was carried out at
paracetamol and salicylic acid, respectively. A Phenomenex
the semicontinuous mode to establish the corresponding optimal
Gemini-NX C18 column (5 lm, 250 mm 4.6 mm) was used for
doses of each coagulant. Furthermore, verification tests were car-
the separation. The mobile phase consisted of a mixture of acetoni-
ried out to confirm if the age of the culture had any influence in
trile:water (30:70, v/v) for the analysis of paracetamol and a mix-
the flocculation behavior. For this purpose, the two most efficient
ture of acetonitrile:water:orthophosphoric acid (70:30:0.1, v/v/v).
flocculants and doses at the steady state were tested with the bio-
HPLC quality acetonitrile (CH3CN) from LAB-SCAN, orthophospho-
mass harvested at the end of a batch culture of 4 days age follow-
ric acid (H3PO4) from Panreac and ultrapure water obtained by a
ing the steady state. Finally, the best flocculant and doses obtained
Millipore System were used for the preparation of the mobile
determined at the semicontinuous mode, were assayed with the
phase. Before use, each mixture was passed through a Millipore
biomass harvested in experiments on the removal of paracetamol
0.45 lm pore size filter and degasified in an ultrasound bath dur-
and salicylic acid to test the influence of these drugs in the coagu-
ing 30 min. For the chromatographic analysis, the mobile phase
lation–flocculation process. All experiments were run in triplicate,
flow rate was 1 ml min1 and the injection volume was 50 lL.
under room temperature and at the pH value of the culture med-
Before analysis, all the samples were centrifuged twice at
ium (7.5 ± 0.5).
7500 rpm for 10 min (SIGMA 2-16P centrifuge).
2.3.2. Analytical methods

2.2.3. Statistical analysis
Biomass concentration was measured by optical density at
Growth kinetics were resolved in OriginPro 8, using the logistic
680 nm by a UV/visible spectrophotometer (BECKMAN DU640)
model, a sigmoidal curve model which describes the relationship
Biomass recovery was calculated from the biomass concentration
between a microorganism’s growth and density in limited environ-
ratio at the clarified zone (Cbt) versus the culture concentration
mental conditions Eq. (2):
at the beginning of the experiment (Cbi), Eq. (3):
K
N¼ ð2Þ Cbt
1 þ eart Recovery of biomass ¼ 1 100 ð3Þ
Cbi
1 1
where N (g l ) is the algal density at time t (h), K (g l ) is the car-
rying capacity (the maximum algal density reached in the culture),
a is a constant in the logistic model which indicates the relative 3. Results and discussion
position from the origin, and r (d1) is the specific growth rate.
Removal kinetics were also resolved in OriginPro 8, using the 3.1. Removal of pharmaceuticals and nutrients
logistic model.
Then, the software was also used to perform an analysis of vari- The microalgae growth curves and their fittings to the logistic
ance, of growth and removal kinetic parameters, using as mean kinetic model, including the positive control and the two treat-
comparison the Fisher LSD test. ments with drugs, are represented as values of biomass versus
59
Respect to microalgae growth rate r, there were significant differ-

ences (p 6 0.05) between the two treatments with drugs. On the
other hand, daily dilutions rates in the semicontinuous culture pro-
duced instability. As a consequence, the growth rate declined
throughout several days until parameters remained constant dur-
ing the steady state at lower values of biomass concentration. In
spite of the higher growth in the batch mode, a larger the biomass
concentration decline was observed for PC and SaC, as compared
with C+. This larger decline may be consequence from an extra
instability in the culture caused by the semicontinuous input of
drugs.
Growth rates obtained in this research were quite higher (above
0.93 day1 for C+, 0.96 day1 for PC and 0.76 day1 for SaC)
(Table 1), than those reported by Kumar et al. (2014) for the same
Fig. 1. Growth curves of Chlorella sorokiniana for: C+ d, PC N and SaC . Dots specie (0.1 day1). Domínguez Cabanelas et al. (2013), described
correspond to experimental data and continuous lines correspond to fittings by the growth rates for C. vulgaris below 0.38 day1 with different domes-
logistic kinetic model during batch culture. Note: experimental points obtained tic wastewaters and a maximum biomass of 1.52 g l1.
during semicontinuous culture are connected with dashed lines.
Nevertheless, Arbib et al. (2013), reported growth rates for
Scenedesmus obliquus close to 1 day1.
time in Fig. 1. All the treatment showed a typical batch growth To evaluate the potential of C. sorokininana as bioremediation
with an exponential phase of about 5 days. The differences among system, the nutrient uptake in each reactor was evaluated. The
the treatments were analysed according to growth kinetic parame- efficiencies in the removal of nitrates and phosphates by the
ters (Table 1). There were no significant differences (p > 0.05) microalgae are displayed in Fig. 2. At the end of the batch culture,
respect the parameter a, which refers to the relative position from nitrates and phosphates removal was close to 100% since the end of
the origin. Therefore, all the treatments revealed the same beha- the exponential growth phase was caused by the depletion of
viour in the lag phase. The end of the exponential growth phase nutrients. At this stage, the removal rates were significantly differ-
was reached in 8 days with C+ and PC and in 9 days with SaC. As ent (p 6 0.05) between SaC and C+. Then, at the steady state, aver-
it can be seen, the treatments with drugs achieved higher biomass age values (Fig. 2) showed that phosphates, with removal rates
concentration than the positive control, the parameter K being sig- higher than 89% for C+ and 99% for the treatments with drugs, were
nificantly different (p 6 0.05) from that of the control. These results more efficiently removed than nitrates. The experimental data in
may be explained by the fact that both drugs were an additional semicontinuous culture revealed significant differences between
source of organic carbon (besides injected CO2) and it is well SaC and C+ in the removal of nitrates; and among the two treat-
known that the genus Chlorella can have a mixotrophic growth. ments with drugs and C+ in the removal of phosphates.
Table 1
Logistic model kinetic parameters (K, a, r) of Chlorella sorokiniana growth and experimental data of their growth (Cbo, Nco, Cbm, Ncm).
C+ PC SaC
Cbo (g l1) 0.0356 ± 0.0000 0.0356 ± 0.0000 0.0356 ± 0.0000
Nco (cell ml1) 3.20 106 ± 0.00 3.20 106 ± 0.00 3.20 106 ± 0.00
Cbm (g l1) 1.4100 ± 0.2907 2.0465 ± 0.0342 2.0545 ± 0.1507
Ncm (cell ml1) 2.12 108 ± 4.88 107 4.20 108 ± 2.17 107 3.15 108 ± 8.48 106
K (g l1) 1.4039 ± 0.2923 2.0901 ± 0.0224 2.1350 ± 0.1318
a 3.7749 ± 0.0053 4.3349 ± 0.2070 4.1589 ± 0.4765
r (d1) 0.9395 ± 0.0572 0.9627 ± 0.0650 0.7679 ± 0.1151
R2 0.9935 0.9939 0.9912
Abbreviations: Cbo = initial biomass; Nco = initial number of cells; Cbm = maximum biomass; Ncm = maximum number of cells; K = carrying
capacity; a = constant of logistic kinetic model; r = microalgae growth rate; R2 = correlation coefficient.
Fig. 2. Volumetric efficiencies of Chlorella sorokiniana in the removal of nitrates and phosphates in batch and semicontinuous culture.
60
Table 2
Volumetric efficiency and specific efficiency in the removal of nitrates and phosphates by Chlorella sorokiniana in the steady state of a semicontinuous culture.
C+ PC SaC
1 1
Volumetric efficiency (mg l d ) NO
3 159.43 ± 9.82 167.04 ± 12.31 180.25 ± 3.46
PO3
4 14.65 ± 0.12 16.33 ± 0.03 16.34 ± 0.02
Specific efficiency (mg g biomass1 d1) NO
3 153.24 ± 12.08 142.86 ± 8.68 216.56 ± 12.82
PO3
4 14.11 ± 0.40 13.98 ± 0.26 19.59 ± 0.86
The obtained results may be explained by the nitrogen and

phosphorus ratio (N:P). The general microalgae composition was
determined by the Redfield ratio (C106H181O45N16P) (Redfield,
1958) and therefore, a N:P ratio of 16:1 is required for an optimal
growth. However, the culture medium used in the experiments
(Mann and Myers, 1968) has a ratio of 9.3:1 so, theoretically, has
a limitation in nitrogen. The ratio remained close to 9 at the end
of the batch culture and an efficient simultaneous elimination of
nitrogen and phosphorus took place. These results are in concor-
dance with those obtained by Arbib et al. (2013), who reported that
for an efficient simultaneous elimination of nutrients, a N:P ratio
between 9 and 13 is required for S. obliquus. They reached 95%
nitrogen removal and 100% phosphorus removal for the optimal
ratio. In the semicontinuous culture the N:P ratio increased over
time due to the higher removal efficiency of phosphate and the
daily addition of fresh medium, which caused an increase in the
relation N:P up to a value of 26. In this case, there was a limitation Fig. 3. Volumetric efficiency in the removal of paracetamol and salicylic acid of
Chlorella sorokiniana during batch culture. Dots correspond to experimental data
of phosphorus, as occurs in STPs, and therefore nitrates removal
and continuous lines correspond to fittings by the logistic kinetic model.
was not complete.
Average values of removal rates of the steady state are
expressed as volumetric performance and specific performance in Table 3
Table 2, for an initial concentration of 729.53 mg l1 of nitrates Logistic model kinetic parameters (K, a, r) of Chlorella sorokiniana for the removal of
pharmaceuticals in the batch culture. Volumetric efficiency and specific efficiency in
and 54.53 mg l1 of phosphates. The obtained results confirm that
the steady state of the semicontinuous culture.
microalgae may be applied for the removal of nutrients removal
from wastewaters due to their high removal rates. However, it is PC SaC
necessary to mention that secondary-influent wastewater has val- K (mg l )
1
17.6168 ± 0.9108 17.6789 ± 0.9589
ues much lower, with a typical concentration of ammonia nitrogen a 4.4924 ± 0.2380 10.2002 ± 3.1565
of 20–40 mg l1 and phosphorus of 1–10 mg l1 (McGinn et al., r (d1) 1.0143 ± 0.0551 4.0668 ± 1.2052
R2 0.9941 0.9919
2011). Nevertheless, several researchers (Aslan and Kapdan, 3.1309 ± 0.2158 6.9806 ± 0.3097
Volumetric efficiency (mg l1 d1)
2006; Arbib et al., 2013; Domínguez Cabanelas et al., 2013; Specific efficiency 2.6836 ± 0.2587 8.3440 ± 1.2060
Gómez et al., 2013) have proven the feasibility of microalgae (mg g biomass1 d1)
production using the effluent from the different stages at STPs. Abbreviations: K = carrying capacity; a = constant of logistic kinetic model;
Results obtained by these researchers demonstrate that the r = pharmaceutical removal rate; R2 = correlation coefficient.
biomass productivity is compatible with removal of nutrients from
wastewater. with values 2.3 times greater for the salicylic acid, reaching their
In order to determine the removal of pharmaceuticals, the daily maximum removal capacity with only 4 days of culture time. In
concentration of paracetamol in PC and that of salicylic acid in SaC any case, at the end of the batch culture, efficiencies above 67%
reactors were compared to the concentrations of these pharmaceu- for paracetamol and 73% for salicylic acid were achieved.
ticals in the corresponding C-. For both pharmaceuticals it was no Furthermore, the relation between the growth kinetics and
observed any decrease of concentration in the respective C-. On the pharmaceuticals removal kinetics in the batch culture was studied.
contrary, paracetamol and salicylic acid content decrease through- There was a constant correspondence for the removal of paraceta-
out time in PC and SaC reactors, respectively. This decrease was mol respect the biomass concentration (8.27 ± 0.54 mg of paraceta-
associated to the removal by C. sorokiniana microalgae. The mol g1 biomass), but there was an exponential increase in the case
removal curves of paracetamol and salicylic acid and the of salicylic acid (up to 53.92 mg of salicylic acid g1 biomass).
corresponding fittings to the logistic kinetic model for the batch On the other hand, the average volumetric efficiencies for the
culture, are showed in Fig. 3. The removal curves display a similar pharmaceuticals removal in the steady stage of the semicontinu-
trend than growth curves, since the removal rates increase is ous culture were 93 ± 2.25% for salicylic acid and 41.75 ± 1.01%
related with the biomass concentration increase. The removal dif- for paracetamol. Therefore, the volumetric efficiency for salicylic
ferences between the two drugs assayed were analysed according acid was 2.2 times higher than for paracetamol.
to the kinetic parameters of the logistic model (Table 3). There No prior studies about pharmaceuticals removal by microalgae
were significant differences (p 6 0.05) respect the parameter a, were found and the information available about other microorgan-
which indicated that the beginning of the removal of salicylic acid isms is scarce. Bacterial biodegradation of paracetamol has been
had a delayed response. Despite the maximum capacity of removal studied by several authors (Zhang et al., 2013; Wu et al., 2012;
did not revealed significant differences (p > 0.05), the removal De Gusseme et al., 2011), achieving high or complete degradations,
kinetic was much quicker for the salicylic acid than for paraceta- even at initial concentration of 2500 mg l1 (70 h) by a species of
mol. The removal rates revealed significant differences (p > 0.05), genus Pseudomonas (Zhang et al., 2013). On the other hand,
61
Combarros et al. (2014) claimed that the biodegradation of salicylic the culture affected the efficiency of flocculation. Differences in
acid by Pseudomonas putida achieved an efficiency of 100% (8 h) the recovery values may be explained by the dependence on the
and 94.7% (24 h) for 100 and 500 mg l1, respectively. composition of the cell wall, the age of the culture, the extent
and type of excretions, physiological conditions and other factors
3.2. Coagulation–flocculation (Avnimelech et al., 1982). Furthermore, the biochemical com-
position of the cell surface is known to vary between exponential
Natural sedimentation of C. sorokiniana was practically non- and steady state cultures, resulting in different flocculation beha-
existent, even with incubation times greater than 60 min (results viour (Danquah et al., 2009; Henderson et al., 2008). In addition,
not shown). This is due to a combination of the small cell size of microalgae excrete organic matter, such as polysaccharides and
the microalgae (3–5 lm), their low concentration in the culture proteins, which are accumulated into the growth medium and
medium (0.5–2.5 g l1), and the electrostatic repulsive forces can compete with the algal surface for the flocculants (Zhang
among cells (Grima et al., 2003). Therefore, their sedimentation et al., 2012; Chen et al., 2009). Chemical flocculation with metal
velocity is lower than 106 m s1 which leads to a non-efficient salts (Zn2+, Al3+ and Fe3+) has been extensively studied and has
process (Granados et al., 2012). been reported that aluminium salts usually have better removal
Flocculation results for metal salts are shown in Fig. 4. It can be rates than iron salts (Papazi et al., 2010). On the other hand, ferric
observed that AlCl3 presented higher efficiencies than FeCl3 for the salts caused changes in the colour of the cells with high concentra-
same dose. Efficiencies higher than 95% were achieved (1 min tion dose and gave yellowish colour to the supernatant, as shown
incubation time), with the biomass harvested at the end of batch by Papazi et al. (2010). Published studies show lower efficiencies
culture (Fig. 4a and b). The doses that proved to be more efficient than those we achieved, neither in the batch culture not in the
were 200 mg g1 for AlCl3 (95.23 ± 0.51%, 1 min) and 250 mg g1 semicontinuous culture. Efficiencies lower than 90% in the recov-
for FeCl3 (98.35 ± 0.46%, 1 min) (Figs. S1 and S2 in supplementary ery of Chlorella minutissima, biomass harvested in a batch culture,
data). In any case, incubation times higher than 15 min led to an with 500 mg l1 of AlCl3 and FeCl3 for a sedimentation time of
improvement of the recovery below 5%. On the other hand, recov- 200 min and a biomass concentration of 2.20. 108 cells ml1
ery efficiencies of the biomass harvested in the steady state are (Papazi et al., 2010). Rakesh et al. (2014) reported efficiencies
shown in the Fig. 4c and d. The aluminium salt provided a recovery below 70% in the recovery of 2 week batch culture of C. sorokiniana
of 88.44 ± 1.47% for 200 mg g1 and the iron salt 86.31 ± 1.43% for with 200 mg l1 of aluminium sulphate and below 85% with
250 mg g1 (60 min incubation time). These recovery values are 200 lM of ferric chloride. Meanwhile, Granados et al. (2012)
lower than those achieved with the same dosage in the batch stage, claimed a low recovery efficiency of 30% with 20 mg l1 of iron
when both salts showed performances higher than 98%. Then, fol- chloride and lower than 15% with 70 mg l1 of aluminium sulphate
lowing the steady state, the verification tests that were next car- in the recovery of Muriellopsis sp. (2.0 g l1 biomass concentration,
ried out with the aluminium salt showed efficiencies higher than 15 min incubation time) from a semicontinuous culture.
90% (15 min incubation time) for the biomass harvested after Flocculation results achieved by synthetic polyelectrolytes are
4 days of batch. Therefore, it may be concluded that the age of displayed in Fig. 5. As it may be seen, best efficiencies were
Fig. 4. Recovery efficiency of Chlorella sorokiniana biomass at the end of a batch culture with different doses of AlCl3 (4a) and FeCl3 (4b) and the recovery efficiency in the
steady state with AlCl3 (4c) and with FeCl3 (4d). The doses are expressed as mg of coagulant per g of biomass.
62
Fig. 5. Recovery efficiency of Chlorella sorokiniana biomass at the end of a batch culture with different doses of CH-35 (5a) and CH-15 (5b) and the recovery efficiency in the
steady state with CH-35 (5c) and CH-15 (5d). The doses are expressed as mg of coagulant per g of biomass.
obtained with the strong cationic polymer CH-35. At the end of the in the batch process (Fig. 6a) (Fig. S7 in supplementary data)
batch culture, efficiencies higher than 99.5% were achieved with showed that recoveries provided by chitosan were as low as
15 mg g1 of CH-35 and 98% with 20 mg g1 of CH-15 (1 min around 15%, which is in agreement with the low recoveries
incubation time) (Fig. 5a and b) (Fig. S3 and S4 in supplementary reported by other researchers (Granados et al., 2012). Sirin et al.
data). Similarly to the metal salts, incubation times longer than (2012) described that alkaline pH is necessary for efficient floccula-
15 min only supposed an improvement of 1% in the recovery. tion process with chitosan. In addition, it has been claimed that
This is consequence that the use of synthetic polyelectrolytes gen- auto-flocculation can be achieved with alkaline pH by adding
erated a single big floc immediately after the addition of the coagu- NaOH (Chen et al., 2011). This is explained by the fact that some
lant (Fig. S5 in supplementary data). On the other hand, recovery chemical ions can precipitate under alkaline conditions with the
efficiencies of the biomass harvested in the steady state are shown algal biomass and favour the flocculation process (Sirin et al.,
in the Fig. 5c and d. Also, as for metal salts, the recovery perfor- 2012). For this reason, in the assays with the harvested biomass
mance was lower than in the batch process, attaining, at the steady in steady state (Fig. 6b), the two best doses of the previous stage
state, efficiencies of 60.96% for 15 mg g1 CH-35 and 84.54% for were tested under pH 10, which was induced by the addition of
25 mg g1 CH-15. Regarding, the verification tests that were car- NaOH 1 N. Moreover, a chitosan dose of 500 mg g1 and then an
ried out with CH-35 following the steady state, after 4 days of induced pH 10 in the absence of chitosan were tested. The best
batch culture showed efficiencies higher than 90% (15 min incuba- results were obtained with 200 mg g1 dose under inducted pH
tion time). Again, the age of the culture was proved to affect the 10 which allowed for a biomass recovery of 69.21% with 15 min
efficiency of flocculation. Despite of their high and quickly effi- incubation time. The combination of chitosan and alkaline pH pro-
ciency in flocculation process, a limitation was detected with the vided better results than both factors separately. These results
floc viscosity, which caused cell aggregates that adhered to the agree with those by Rakesh et al., 2014, who reported the effect
wall of the test tubes (Fig. S6 in supplementary data). This aggrega- of alkaline pH values of 10 and 11 were effective for the most of
tion constitutes an important limitation for the retrieval of the bio- the strains tested but not showed any significant change for C.
mass, so impeding its subsequent use. Granados et al. (2012) sorokiniana MIC-G5. However, it has been described that 8.5–10.5
claimed that efficiencies higher than 90% were achieved in the pH values allow microalgae recovery rates above 90% with
recovery of Muriellopsis sp. (biomass harvested in a semicontinu- Phaeodactylum tricornutum (Sirin et al., 2012) or Anabaena marina
ous culture), with only 10 mg l1 of cationic polymer (EM 16), (González López et al., 2009). On the other hand, autoflocculation
which was characterized by medium charge density and med- with induced pH by cellular growth (without CO2 control), were
ium-large molecular weight (2.0 g l1 biomass concentration, assayed due to the positive previous results with alkaline pH.
15 min incubation time). The used of cationic polymers has been After a period of 2 days, 9.6 pH was reached and 200 mg g1 dose
previously reported for the microalgae flocculation although, the showed efficiencies below 20% (results not shown). These low
optimum doses depend on the nature of the polymer. results may be explained by the fact that autoflocculation is asso-
Flocculation results obtained with chitosan are represented in ciated with the formation of calcium and magnesium precipitates,
Fig. 6. For the tested doses, the results with the biomass harvested which can carry positive surface charges and may induce
63
Fig. 6. Recovery efficiency of Chlorella sorokiniana biomass at the end of a batch culture (6a) and in a steady state (6b) with different doses of chitosan. The doses are
expressed as mg of coagulant per g of biomass.
flocculation by charge neutralization. Calcium phosphate precipi- was faster removed from water than paracetamol in the batch cul-
tates are positive charged, but it is necessary calcium ions in excess ture. Then, in the semicontinuous culture the volumetric efficiency
and high phosphate concentrations (Vandamme et al., 2013). was higher for salicylic acid than for paracetamol. Finally, the floc-
However, in this work, due to the nutrients removal process asso- culants here used resulted in different efficiencies and flocs charac-
ciated to the microalgae growth, the level of nutrients at the end of teristics, which must be considered in the flocculant of choice
the batch process and at the steady state was low. Indeed, the con- depending on the subsequent use of the biomass.
centration of phosphates had been reduced by a 90% at the
moment when flocculation tests were carried out (Fig. 2). Acknowledgements
Eventually, considering 200 mg g1 AlCl3 as the most effective
flocculant and dose among all tested, it was used for the floccula- This research was supported by Spanish Ministry of Educations,
tion of the biomass harvested in the experiments on the removal of Culture and Sports (FPU12/03073), University of León (doctorate
drugs. The results did not reveal significant differences (p > 0.05) research program ULE-2014) and Spanish Ministry of Economy
between the positive control and the treatments with drugs, nei- and Competitiveness, State Secretariat for Research, Development
ther with the results already obtained in the previous flocculation and Innovation (RYC-2010-05634).
assays (Fig. 4a and c). The flocculation results of biomass harvested
at the end of the batch process showed efficiencies above 99%.
Likewise, the results with the biomass harvested in the steady state Appendix A. Supplementary data
showed efficiencies close to 90%, which were lower than for the
batch stage. Therefore, it can be assumed that the presence of Supplementary data associated with this article can be found, in
drugs in the water to be treated did not affect the AlCl3 flocculant the online version, at http://dx.doi.org/10.1016/j.biortech.2015.03.
efficiency. The flocculants nature and their concentration are 004.
determining factors in the efficiency of the process. Apart from
the flocculants price, depending on the destination of the microal- References
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65
SUPPLEMENTARY DATA

Fig. S1 Flocculation with AlCl3 with 1 hour incubation time at 100, 150,200, 250 and 300 mg g‐1
(from left to right)

Fig. S2 Flocculation with FelCl3 with 1 hour incubation time at 100, 150,200, 250 and 300 mg g‐1
67

Fig. S3 Flocculation with CH‐15 with 1 hour incubation time at 2.5, 5, 10, 15 and 20 mg g‐1 (from left
to right)

Fig. S4 Flocculation with CH‐35 with 1 hour incubation time at 2.5, 5, 10, 15 and 20 mg g‐1 (from left
to right)
68

Fig. S5 Single big floc generated immediately after the addition of a synthetic polyelectrolyte

Fig. S6 Detail of the floc viscosity after the addition of a synthetic polyelectrolyte, which caused cell
aggregates that adhered to the wall of the glass during the previous stirring
69

Fig. S7 Flocculation with chitosan with 1 hour incubation time at 20, 40, 100, 150 and 200 mg g‐1

Fig. S8 Flocs generated after the addition of FeCl3, AlCl3, chitosan, CH‐35 and CH‐15 (from left to
right)
70

Capítulo 5
Artículo II
Paracetamol and salicylic acid removal from contaminated
water by microalgae

Journal of Environmental Management, (2016) doi: 10.1016/j.jenvman.2016.06.051
Journal of Environmental Management xxx (2016) 1e8
Journal of Environmental Management

journal homepage: www.elsevier.com/locate/jenvman
Research article
Paracetamol and salicylic acid removal from contaminated water by

microalgae*
C. Escapa, R.N. Coimbra, S. Paniagua, A.I. García*, M. Otero*
n, 24071 Leo
Department of Applied Chemistry and Physics, Institute of Environment, Natural Resources and Biodiversity (IMARENABIO), University of Leo n,
Spain
Article history: The biomass growth, pharmaceutical removal and light conversion efficiency of Chlorella sorokiniana
Received 18 November 2015 under the presence of paracetamol (PC) and salicylic acid (SaC) were assessed and compared at two
Received in revised form different concentrations of these pharmaceuticals (I: 25 mg l1, II: 250 mg l1). Microalgae were resistant
15 June 2016
to these concentrations and, moreover, their growth was significantly stimulated (p 0.05) under these
Accepted 25 June 2016
Available online xxx
drugs (biomass concentration increased above 33% PCI, 35% SaCI, 13% PCII and 45% SaCII, as compared
with the respective positive controls). At the steady state of the semicontinuous culture, C. sorokiniana
showed removal efficiencies above 41% and 69% for PCI and PCII, respectively; and above 93% and 98% for
Keywords:
Chlorella sorokiniana
SaCI and SaCII, respectively. Under an irradiance of 370 mE m2 s1, higher quantum yields were reached
Pharmaceutical industry by microalgae under the presence of drugs, either at dose I or II, than by the respective positive controls.
Wastewater These results point to C. sorokiniana as a robust strain for the bioremediation of paracetamol and salicylic
Emerging contaminants acid concentrated wastewaters.
Bioremediation © 2016 Elsevier Ltd. All rights reserved.
Phycoremediation
1. Introduction treatment plants (STPs), which is probably the most investigated

route of entry. Still, some authors (Larsson et al., 2007; Carlsson
The global increase of the production and use of drugs has led to et al., 2009; Larsson and Fick, 2009) have pointed to the highly
a growing international awareness on the disposal of pharmaceu- concentrated effluents from drugs production facilities as impor-
tical wastes due to their potential deleterious effects (Halling- tant sources of pharmaceuticals to the environment. These
Sorensen et al., 1998). In the aquatic environment, the presence concentrated effluents should be appropriately treated to remove
of pharmaceuticals may cause physiological responses in organisms active pharmaceuticals ingredients (APIs) before discharge, either
for which they were not intended, such as accumulation in tissues, in natural waters or in the municipal sewage system.
reproductive damage, inhibition of cell proliferation and behav- To date, the nonexistence of limiting regulations (Bolong et al.,
ioural changes (Escher et al., 2011). 2009; Barcelo and Petrovic, 2006) has led to a lack of control on
Pharmaceuticals are known to reach the environment through the discharge of APIs by manufacturing facilities (Larsson and Fick,
usage and/or inappropriate disposal (Khetan and Collins, 2007). 2009). However, given public concern about the presence of phar-
Therefore, main sources of pharmaceuticals in the environment maceuticals in the environment, it is expectable that regulations
include hospitals (Gupta et al., 2009; Sua rez et al., 2009; Escher will come out in the near future. Yet, conventional wastewater
et al., 2011; Verlicchi et al., 2012), landfill leachates (Andrews treatments were designed to eliminate carbon, nitrogen and
et al., 2012; Clarke et al., 2015) and effluents from sewage phosphorus, but not to remove APIs, which pose high biological
activity at low concentrations, are mostly hydrophilic and have low
adsorption rates (Martz, 2012).
Advanced oxidation processes (AOPs) such as oxidation, ozon-
*
This work was presented (and selected for possible publication by JEMA) at the
ation, perozonation, direct photolysis, TiO2 photocatalysis, solar
IWWATV Conference, which was held in Athens (Greece) from 21st to 23rd of May,
2015. photocatalysis, Fenton reactions and ultrasonic irradiation have
* Corresponding authors. been pointed as promising treatments for the removal of APIs from
E-mail addresses: carla.escapa@unileon.es (C. Escapa), ricado.decoimbra@ wastewater (Deegan et al., 2011). Nevertheless, the high opera-
unileon.es (R.N. Coimbra), spanb@unileon.es (S. Paniagua), ana.garcia@unileon.es tional costs, the harsh reaction conditions and the generation of
(A.I. García), marta.otero@unileon.es (M. Otero).
http://dx.doi.org/10.1016/j.jenvman.2016.06.051
0301-4797/© 2016 Elsevier Ltd. All rights reserved.
73
2 C. Escapa et al. / Journal of Environmental Management xxx (2016) 1e8
secondary pollutants are important disadvantages of this sort of medium, the PBRs and the tubing for the supply of air were ster-
treatments (Wu et al., 2012). Conversely, biodegradation treat- ilized in autoclave (121 C, 1 atm, and 20 min) to avoid contami-
ments are considered as a low-cost and environmentally friendly nations. In the experiments under the presence of paracetamol (PC)
option (Hasan et al., 2011; Chen et al., 2010). Although there are or salicylic acid (SaC), these drugs were added to the sterilized
preliminary studies about biodegradation of pharmaceuticals by culture medium but not submitted to the sterilization process so to
microorganisms (Combarros et al., 2014; Zhang et al., 2013; Wu guarantee their stability.
et al., 2012; De Gusseme et al., 2011), very few studies have been
carried out on the removal of pharmaceuticals by microalgae 2.2. Experimental set-up
(Gupta et al., 2015 and references within). On the contrary,
microalgae capacity to remove nutrients from wastewater is well PBRs were operated in batch mode until the end of the expo-
known, Choudhary et al. (2016) having highlighted the importance nential growth phase. Then, the operation mode was semi-
of the selection of robust strains/consortia that can efficiently grow continuous with a daily renewal rate of 30%. Therefore, under
under concentrated wastewaters. However, to our best knowledge, semicontinuous operation, 30% of the reactor culture volume was
there are not published results on microalgae resistance and effi- daily taken off and replaced by fresh medium (containing 25 mg l1
ciency under extreme concentrations of drugs, which may be ex- of either paracetamol or salicylic acid, in the case of PC and SaC
pected in wastewaters from pharmaceutical industry. treatments, respectively). PBRs were operated in semicontinuous
In this context, and after having proved the capability of Chlor- mode until stability of the growth parameters at the steady state.
ella sorokiniana to remove pharmaceuticals from water (Escapa The experiments carried out consisted of inoculated culture
et al., 2015), the aim of this work was to assess microalgae medium together with paracetamol (PC) or salicylic acid (SaC). Two
response towards relatively high drugs concentration. For this different and non-simultaneous sets of experiments were run un-
purpose, microalgae growth kinetics, pharmaceutical removal rates der two different concentrations of these pharmaceuticals: (i)
and light conversion efficiency under paracetamol and salicylic acid dosage I (25 mg l1), as described by Escapa et al. (Escapa et al.,
were evaluated and compared at two different concentrations of 2015), which were named as PCI and SaCI; and (ii) dosage II
these drugs, which are among the most consumed and found in (250 mg l1), named as PCII and SaCII, respectively. For each set of
natural waters worldwide. In the aquatic environment, paraceta- experiments, positive controls with inoculated culture medium
mol is easily accumulated due to its relative high solubility and without drugs (CþI and CþII, respectively) were run in parallel.
hydrophilicity (Muir et al., 1997). Regarding salicylic acid, its Also, negative controls with not inoculated culture medium but PC
toxicity and tendency to accumulate in the environment are well or SaC were run simultaneously with each concentration set of
known (Combarros et al., 2014). Therefore, results obtained in this experiments (CP-I, CSa-I and CP-II, CSa-II, respectively). The treat-
work are expected to contribute to the knowledge on the applica- ments and the abbreviations used in this work are displayed in
tion of microalgae on the bioremediation of wastewater, namely on Table s1 (as supplementary material). Three simultaneous repli-
the removal of emerging contaminants such as pharmaceuticals. cates of each treatment were operated under identical conditions
for each set of experiments. Paracetamol (C8H9NO2, 99%) was
2. Materials and methods supplied by Roic Pharma and salicylic acid (C7H6O3, 99%) by
Panreac. Physico-chemical properties of these pharmaceuticals are
2.1. Microorganism and culture conditions displayed in Table s2 (as supplementary material).
Throughout the experiments, the culture growth was daily
The microalgae strain used in this study was Chlorella sor- monitored by biomass concentration and cell density. Also, to
okiniana CCAP 211/8 K (UTEX Culture Collection). Inoculum for the assess the pharmaceuticals removal, the concentration of paracet-
experiments was cultivated in the standard culture medium Mann amol and salicylic acid was daily monitored. In addition, the irra-
and Myers (Mann and Myers, 1968) in 250 ml Erlenmeyer flasks. diance in the absence of cells in the central point inside of the PBR
The culture medium is composed of (grams per litre of distilled was measured to evaluate the light conversion efficiency of the
water): 1.2 MgSO4$7H2O, 1.0 NaNO3, 0.3 CaCl2, 0.1 K2HPO4, cells.
3.0 102 Na2EDTA, 6.0 103 H3BO3, 2.0 103 FeSO4$7H2O,
1.4 103 MnCl2, 3.3 104 ZnSO4$7H2O, 7.0 106 Co(N-
2.3. Analytical methods
O3)2$6H2O, 2.0 106 CuSO4$5H2O. The C. sorokininana inoculum
was maintained inside a vegetal culture chamber, where the
Biomass concentration (Cb) was determined by optical density at
growth conditions remained constant, under controlled tempera-
680 nm (OD680) by spectrophotometric (UV/visible spectropho-
ture (25 ± 1 C), irradiance (175 mE m2 s1), photoperiod (12:12)
tometer BECKMAN DU640) and verified by dry weight. Preliminary
and stirring (250 rpm).
studies were conducted to determine OD680 as a function of dry
Experiments were carried out in bubbling column photo-
weight as shown in Eq. (1):
bioreactors (PBRs) with spherical bases (40 mm diameter and
300 mm height with 300 ml capacity), keeping an operating vol-
OD680 ¼ 5:1834 Cb þ 0:0128; R2 ¼ 0:9983 (1)
ume of 250 ml. PBRs containing Mann and Myers medium were
inoculated with the above inoculum of C. sorokiniana in order to For the determination of dry weight, 10 ml of culture were
have an initial microalgae concentration of about 3.2 106 cells filtered through a 0.45 mm Whatman filter, which was then washed
ml1. Then, culture conditions were kept constant under controlled with 20 ml HCl (0.5 M) to dissolve precipitated salts, and dried in an
temperature (25 ± 1 C), irradiance (370 mE m2 s1) and photo- oven at 80 C for 24 h. Dry weight was then obtained by mass
period (12:12), inside a vegetal culture chamber. PBRs were illu- difference of the filter after and before filtration. Additionally, the
minated by 8 fluorescent lamps (58 W, 2150 lumen, Philips, France) growth of the culture was measured as cell density (Nc) by cell
and aerated at a rate of 0.3 v/v/min with CO2 enriched at 7% v/v, counting with a Neubauer chamber.
which was injected on demand to keep a constant pH A Waters HPLC 600 equipped with a 2487 dual l absorbance
(pH ¼ 7.5 ± 0.5) as controlled by a pH sensor. Before injection, the detector was used for the quantification of the target pharmaceu-
air was filtered through 0.2 mm sterile air-venting filter (Millex- ticals. The wavelengths of detection were 246 and 236 nm for
FG50, Millipore). At the beginning of the experiments, the culture paracetamol and salicylic acid, respectively. A Phenomenex C18
74
C. Escapa et al. / Journal of Environmental Management xxx (2016) 1e8 3
column (5 mm, 250 mm 4.6 mm) was used for the separation. The state of the semicontinuous culture specific efficiencies were given
mobile phase consisted of a mixture of acetonitrile:water (30:70, v/ as mgremoved g1 1
biomass d .
v) for the analysis of paracetamol and a mixture of acetoni- During the microalgae culture, the biomass extinction coeffi-
trile:water:orthophosphoric acid (70:30:0.1, v/v/v). HPLC quality cient (Ka, m2 g1) was determined by the following equation (Eq.
acetonitrile (CH3CN) from LAB-SCAN, orthophosphoric acid (5)):
(H3PO4) from Panreac and ultrapure water obtained by a Millipore
System were used for the preparation of the mobile phase. Before
use, each mixture was passed through a Millipore 0.45 mm pore size OD400700
Ka ¼ (5)
filter and degasified in an ultrasound bath (30 min). For the chro- Cb r
matographic analysis, the mobile phase flow rate was 1 ml min1
and the injection volume was 50 ml. Before analysis, all the samples where OD400700 is the average optical density in the visible range,
were centrifuged twice at 7500 rpm for 10 min (SIGMA 2e16P which is determined by spectrophotometric analysis (UV/visible
centrifuge). spectrophotometer BECKMAN DU640) and r is the cuvette optical
path (m).
The average irradiance at which cells are exposed inside a cul-
2.4. Data analysis ture (Iav, mE m2 s1) was calculated on the basis of Ka, by the
equation defined by Grima et al (Grima et al., 1997). (Eq. (6)):
For the description, analysis and comparison of the experi-
mental results here obtained, different models and/or parameters
were determined according to the equations that are next depicted. I0
Iav ¼ ð1 expð Ka r Cb ÞÞ (6)
The logistic equation (Eq. (2)) was used for mathematical modelling ðKa r Cb Þ
of microalgae biomass growth in batch culture. This classic model
was originally described by Verhulst (1838) and has been proved to where Io (mE m2 s1) is the irradiance in the absence of microalgae,
fit the growth of microalgae (Xin et al., 2010). Fittings of the and r is the light path inside the reactor (m).
experimental daily values of biomass concentration (N, g l1) The light conversion efficiency may be approached by the
versus time (t, h) to the logistic equation were determined by calculation of the quantum yield (JE, g E1). This represents the
OriginPro8, a scientific graphing and data analysis software. amount of microalgae biomass generated by unit of radiation
(usually a mole of photons) absorbed by the culture and may be
K calculated by the expression defined by Grima et al. (1997) (Eq. (7)):
N¼ (2)
1 þ eart
where the three parameters that have to be estimated are: K (g l1)

Pb
is the carrying capacity (the maximum algal density reached in the JE ¼ (7)
Fvol
culture), a is a constant in the logistic model which indicates the
relative position from the origin and indicates the duration of the
where Pb (g m3 d1) is the volumetric biomass productivity and
lag phase, and r (d1) is the specific growth rate.
Fvol (mE m3 d1) is the photon flux absorbed in the volume unit,
The logistic equation (Eq. (2)) was also used to describe the ki-
which may be calculated by applying Eq. (8) (Grima et al., 1997):
netics of the removal of pharmaceuticals in the batch culture. In this
case, for each pharmaceutical, N is the daily volumetric efficiency
(N, mgremoved l1); K (g l1) is the maximum removal capacity by Fvol ¼ Iav Ka Cb (8)
the microalgae; a is a constant indicating the duration of the lag
phase, that is, the delay in the removal of the target compound; and Considering the quantum yield (JE) defined by Grima et al.
r (d1) is the specific removal rate. OriginPro8 was used to deter- (1997) and aiming to determine the ratio between the removal of
mine the corresponding fittings of the experimental values of the pharmaceuticals and photon flux absorbed, a new equation was set
removal volumetric efficiency (N, mgremoved l1) versus time (t, h). in this work (Eq. (9)). In this equation, the removal yield (JR, g
For each pharmaceutical, the removal volumetric efficiency was removed E1) in microalgae cultures was defined as the amount of
calculated as indicated by the following equation (Eq. (3)): pharmaceutical removed by microalgae by unit of radiation
absorbed by the culture (Eq. (9)):
Volumetric efficiency ¼ Cinf Cefflu D (3)
Rb
where Cinf (mg l1) is the average drug concentration in the JR ¼ (9)
Fvol
influent, Cefflu (mg l1) is the average concentration in the effluent
for each sampling day and D (d1) is the daily dilution rate to each
where Rb (g removed m3 d1) is the volumetric removal efficiency
operation stage. During the batch culture the removal volumetric
and Fvol (mE m3 d1) is the photon flux absorbed in the volume
efficiencies were cumulatively expressed as mg l1. Meanwhile, at
unit.
the steady state of the semicontinuous culture volumetric effi-
In this work, the Mann-Whitney non-parametric test, which
ciencies were expressed as mg l1 d1.
was performed by OriginPro8, was used to test the differences
The specific removal efficiency for each pharmaceutical was
between the treatments here conducted regarding the kinetic pa-
calculated as the ratio between the volumetric efficiency and the
rameters on microalgae growth and pharmaceuticals removal
biomass concentration (Cb, g l1) (Eq. (4)).
during the batch culture, regarding the specific efficiency on the
Volumetric efficiency removal of pharmaceuticals at the steady state of the semi-
Specific efficiency ¼ (4) continuous culture, and regarding the quantum yield and the
Cb
removal yield at the steady state of the semicontinuous culture. The
During the batch culture specific removal efficiencies were significance level was defined at p 0.05 and the p-values are
expressed cumulatively as mgremoved g1
biomass. Then, at the steady depicted as Supplementary Material (Tables s3, s4 and s5).
75
3. Results and discussion After the batch stage, when reactors started to be operated
under semicontinuous mode, a biomass decline occurred for all the
3.1. Biomass growth treatments (Fig. 1a and b). This decline, which is typically associated
to the outset of semicontinuous culture, is related to the instability
The microalgae growth curves are represented in Fig. 1. caused by the daily renewal rate applied. In the case of the PC and
As may be seen in Fig. 1, biomass showed the same typical batch SaC, the biomass decline was more accentuated than for positive
growth curve under all the treatments. Positive controls CþI and controls since the renewal rate also meant a daily addition of either
CþII, which were respectively run in parallel with the dose I and the paracetamol or salicylic acid. Then, after an adaptive lag, during
dose II treatments, showed very similar growth during batch cul- which cells became adapted to the new culture conditions, all
ture. Then, it is evident that the addition of pharmaceuticals treatments reached the steady state under semicontinuous opera-
stimulated microalgae growth during the batch culture as tion. The biomass decline at the beginning of the semicontinuous
compared with positive controls. This may be explained by the fact culture was more evident and the adaptive lag longer for CþII than
that the drugs were an additional source of organic carbon and it is for CþI. Differences between positive controls, which were not run
well known that the genus Chlorella can have a mixotrophic simultaneously, point to a higher instability during the second set
growth. However, while PCI and PCII reached similar maximum of experiments.
biomass concentration at the end of the batch culture (Fig. 1a), in
the case of salicylic acid, larger biomass concentration was attained 3.2. Pharmaceuticals removal
under SaCII than under SaCI (p 0.05) (Fig. 1b). This divergent
behaviour, which was evidenced by the utilization of two different The negative controls C- allowed verifying that neither salicylic
dosages in this work, points to a higher affinity for the consumption acid nor paracetamol showed any decrease of concentration
of salicylic acid by C. sorokiniana. Differences among the treatments throughout the duration of experiments. Therefore, it may be
were analysed according to growth kinetic parameters (Table 1). assumed that the concentration decrease of these pharmaceuticals
With regard to the parameter a, the addition of salicylic acid at the in PC and SaC reactors, may be associated to the removal by
initial pharmaceutical dose II (SaCII) involved the greatest increase C. sorokiniana microalgae.
of the lag phase respect the positive control (CþII) (p 0.05), as The removal curves of paracetamol and salicylic acid during the
shown in Table 1. This delayed response led to a longer exponential batch culture and the corresponding fittings to the logistic kinetic
growth phase for SaCII, which was reached in 12 days, while the model are shown in Fig. 2. These curves displayed a similar trend
rest of the treatments reached this stage within 7e9 days (Fig. 1b). than growth curves (Fig. 1), which points to the relation between
Therefore, it may be said that the growth response of the cells may the pharmaceuticals removal and the increase of biomass
be modified by the addition of pharmaceuticals, which was noto- concentration.
rious for SaCII. Regarding parameters of the logistic model (Table 2), similar
Regarding the carrying capacity (K), it was significantly larger values of the parameter a were determined for PCI and PCII
(p 0.05) under the presence of any of the drugs than for positive (p > 0.05). However, SaCII displayed a significantly higher a value
controls, either at initial dose I or II (Table 1). The K values deter- than SaCI (p 0.05), which denotes that the beginning of the
mined for the two doses of paracetamol (PCI, PCII), although removal of salicylic acid in SaCII had a delayed response as
significantly different, were mostly equivalent. On the other hand, compared with SaCI. This delayed response is related to the delay in
under salicylic acid, K values were significantly higher than under the beginning of the exponential growth in this treatment that was
paracetamol. Moreover, the K determined for SaCII was larger observed in Fig. 1b. Regarding the maximum removal capacity
(p 0.05) than for SaCI. According to properties in Table s2 (in (Table 2), a one order of magnitude higher K was determined under
supplementary material), a main issue may be the distribution of dosage II than I (p 0.05), both for paracetamol and salicylic acid.
the salicylic acid species depending on the pH. For salicylic acid, the Also, although there were no significant differences between PCI
unique species at pH ¼ 7.5 is the anionic form (100%) while neutral and SaCI (p > 0.05), SaCII displayed a significantly higher K than PCII
paracetamol is the most frequent species (99.4%) at this pH. (p 0.05). With regard to the removal rate r, PCI and PCII displayed
Therefore, it may be expected that the anionic salicylic acid is similar values (p > 0.05), as for SaCI and SaCII (p > 0.05) (Table 2). As
completely dissolved and easily available for microalgae. seen in Fig. 2, the maximum salicylic acid removal capacity was
Microalgae growth rate (r) under the initial paracetamol dose II reached within only 4 days of culture time under both initial
(PCII) was larger (p 0.05) than for the positive control (CþII) pharmaceutical doses, as compared to 7e8 days for paracetamol. In
(Table 1). However, under salicylic acid, both SaCI and SaCII showed fact, the r revealed significant differences, SaCI and SaCII displaying
a smaller (p 0.05) r than the respective positive controls. In values 4 and 5 times greater than PCI and PCII, respectively
addition, SaCII revealed a smaller r than SaCI. Therefore, the addi- (Table 2). At the end of the batch culture, efficiencies above 67% and
tion of salicylic acid led to a higher carrying capacity and a slower 48% were achieved for PCI and PCII, respectively. Meanwhile, sali-
growth rate than the paracetamol treatments and these results cylic acid removal efficiency was above 73% for SaCI and above 94%
were more pronounced under the highest dosage. for SaCII at the end of the batch culture.
Growth rates (r) here obtained for the positive controls and The specific efficiency during the batch culture is represented in
under the addition of paracetamol or salicylic acid (Table 1) were Fig. s1 (as supplementary material). In the case of paracetamol,
quite higher than those reported by Kumar et al. (2014) for the either for PCI or for PCII, there was a constant ratio between the
same strain (0.1 day1) cultivated in TAP medium in Erlenmeyer removal of paracetamol and the biomass concentration throughout
flasks under 100 mE m2 s1 irradiation. Domínguez Cabanelas et al. time. Differently, for salicylic acid, both for SaCI and SaCII, showed
(2013), described growth rates for Chlorella vulgaris below 0.38 an exponential increase until the 4th day.
day1 and a maximum biomass of 1.52 g l1 cultivated with The average specific efficiencies on the removal of pharmaceu-
different domestic wastewaters in 2 l borosilicate reactors under ticals at the steady state of the semicontinuous culture are reported
150 mE m2 s1 irradiation. Nevertheless, Arbib et al. (2013), re- in Fig. s2 (as supplementary material). However different biomass
ported growth rates for Scenedemus obliquus close to 1 day1 growth occurred during the semicontinuous culture of the two sets
cultivated in pre-treated urban wastewater in 2 l Pyrex bottles of experiments, specific efficiencies, which are expressed by mass
under 250 mE m2 s1 irradiation. unit of biomass, allow for the comparison of the removal of
76
Fig. 1. Growth curves of Chlorella sorokiniana for paracetamol (CþI , PCI :, CþII -, PCII A) (a) and salicylic acid (CþI , SaCI :, CþII -, SaCII A) (b) under the initial
pharmaceutical doses I (25 mg l1) and II (250 mg l1). Dots correspond to experimental data and continuous lines correspond to fittings by the logistic kinetic model during batch
culture. Experimental points obtained during semicontinuous culture are connected with dashed lines. Notes: Curves corresponding to CþI and CþII are represented both in (a) and
(b) for comparison purpose. Experiments were performed in triplicate and bars show standard derivations.
Table 1
Logistic model kinetic parameters (K, a, r) of Chlorella sorokiniana growth and experimental data of their growth (Cbm, Ncm) for the positive control (Cþ), paracetamol (PC) and
salicylic acid treatments (SaC), for the initials concentrations I and II. Note: n ¼ 3.
Cþ PC SaC
I II I II I II
Cbm (g l1) 1.41 ± 0.29 1.41 ± 0.27 2.05 ± 0.03 1.79 ± 0.14 2.05 ± 0.15 2.75 ± 0.08
Ncm (cell ml1) 2.12 108 ± 4.88 107 2.03 108 ± 4.41 107 4.20 108 ± 2.17 107 1.63 108 ± 6.01 107 3.15 108 ± 8.48 106 3.84 108 ± 5.30 106
a 3.77 ± 0.01 2.96 ± 0.11 4.33 ± 0.21 3.11 ± 0.21 4.16 ± 0.48 4.13 ± 0.27
K (g l1) 1.40 ± 0.29 1.65 ± 0.25 2.09 ± 0.02 1.90 ± 0.10 2.14 ± 0.13 3.02 ± 0.03
r (d1) 0.94 ± 0.06 0.71 ± 0.05 0.96 ± 0.07 0.95 ± 0.13 0.77 ± 0.12 0.54 ± 0.02
R2 0.9935 0.9841 0.9939 0.9812 0.9912 0.9928
Abbreviations: Cbm ¼ maximum biomass; Ncm ¼ maximum number of cells; K ¼ carrying capacity; a ¼ constant of logistic kinetic model; r ¼ microalgae growth rate;
R2 ¼ correlation coefficient.
pharmaceuticals at this stage. In this sense, it may be stated that the about other microorganisms is scarce. Bacterial biodegradation of
salicylic acid removal by unit of biomass was similar (p > 0.05) to paracetamol at different concentrations has been studied by several
the paracetamol removal under dose II (99.62 ± 16.84 mgremoved authors (Zhang et al., 2013; Wu et al., 2012; De Gusseme et al.,
g1
biomass d
1
for SaCII, 101.16 ± 9.01 mgremoved g1 biomass d
1
PCII). 2011), achieving high or complete degradation even at initial con-
Nevertheless the average specific efficiency for the removal of centrations as high as 2,500 mg l1 (70 h) by a species of genus
salicylic acid at dosage I was higher (p 0.05) than for paracetamol Pseudomonas (Zhang et al., 2013). On the other hand, Combarros et
(8.34 ± 0.38 mgremoved g1 biomass d
1
for SaCI, 2.68 ± 0.11 mgremoved al. (2014) claimed that the biodegradation of salicylic acid by
g1
biomass d1
PCI). Moreover, the efficiencies per gram of biomass Pseudomonas putida achieved an efficiency of 100% (8 h) and 94.7%
achieved for dose II were much larger (p 0.05) than for dose I. (24 h) for initial concentrations of 100 and 500 mg l1, respectively.
No information is available on the compared removal of Therefore, although in a different concentration range, these au-
different concentrations of pharmaceuticals by microalgae and data thors (Combarros et al., 2014) verified a decrease of efficiency with
77
Fig. 2. Chlorella sorokiniana volumetric efficiency in the removal of paracetamol (PCI , PCII -) (a) and salicylic acid (SaCI , SaCII -) (b) during batch culture under the initial
pharmaceutical doses I (25 mg l1) and II (250 mg l1). Dots correspond to experimental data and continuous lines correspond to fittings by the logistic kinetic model. Note:
Experiments were performed in triplicate and bars show standard derivations.
Table 2 centrate by the microalgae Selenastrum capricornutum and Chla-

Logistic model kinetic parameters (K, a, r) of Chlorella sorokiniana for the removal of mydomonas reinhardtii. After seven days of culture, these authors
paracetamol (PC) and salicylic acid (SaC) in the batch culture, for the initials con-
centrations I and II. Note: n ¼ 3.
(Hom-Díaz et al., 2015) determined removals above 88% for E2 and
above 60% for EE2.
PC SaC
I II I II
3.3. Light conversion efficiency
a 4.49 ± 0.24 3.98 ± 1.27 10.20 ± 3.16 14.90 ± 0.05
K (mg l1) 17.62 ± 0.91 146.46 ± 41.01 17.68 ± 0.96 234.38 ± 0.69
r (d1) 1.01 ± 0.06 0.90 ± 0.45 4.07 ± 1.21 4.57 ± 0.07 It was hypothesized that the presence of either salicylic acid or
R2 0.9941 0.9657 0.9919 0.9990 paracetamol in the culture medium could modify the light con-
Abbreviations: K ¼ carrying capacity; a ¼ constant of logistic kinetic model;
version efficiency of the cells since the biomass productivity of the
r ¼ pharmaceutical removal rate; R2 ¼ correlation coefficient. microalgae was affected by the addition of these pharmaceuticals.
In order to verify this hypothesis, the development of the light
availability inside the culture and the quantum yield were deter-
the increase of initial concentration, which is contrary to our mined for both initials pharmaceuticals doses. For an extinction
findings. In any case, removal rates by Pseudomonas putida deter- coefficient value of 0.3161 m2 g1, the average irradiance inside the
mined by these authors (Combarros et al., 2014) were larger than PBR (Iav) showed a gradual decrease corresponding to the increase
removal rates determined in this work. Still, the use of microalgae of the biomass concentration, which reduced the irradiance avail-
in wastewater treatment has been proved to have numerous ad- able inside the culture, as may be seen in Fig. 3. The results dis-
vantages. Among them, the recovery of algal biomass offers the played a sharp decline during the first days due to the exponential
possibility of recycling the assimilated nitrogen and phosphorus as biomass growth phase. Then, at the beginning of the semi-
a fertilizer, as a source of products (e.g. paraffin, olefin, glycerol, continuous culture, there was a slight increase of the light available
protein, anti-oxidant, pigment, plastic, etc.), or as biofuel, and also due to the decrease in the biomass concentration caused by the
the generation of oxygenated high-quality effluent into the water destabilization of the culture. Finally, at the steady state of the
body (Matamoros et al., 2016). semicontinuous culture Iav showed a trend to stabilize throughout
Published results on the removal of emerging contaminants by time. Therefore, the average irradiance at which cells were exposed
microalgae have revealed different efficiencies depending on the (Fig. 3) showed an inversely proportional behaviour to the trend of
pollutant and on the microalgae strain. For example, Gattullo et al. biomass growth (Fig. 1). These results are in agreement with those
(2012) demonstrated that Monoraphidium braunii was able to obtained by Go mez et al. (2013) for a Muriellopsis sp. culture, with
remove up to 48% of bisphenol A with an initial concentration of an Iav value of 43 mE m2 s1 under semicontinuous operation with
4 mg l1. de Wilt et al. (2016) reported that C. sorokiniana grown in an Io value of 1850 mE m2 s1, which means an irradiance reduction
wastewater streams was able to achieve removal efficiencies up to of about 97%.
60e100% for the removal of diclofenac, ibuprofen, paracetamol and The quantum yields (JE) at the steady state of the semi-
metoprolol. However, these authors (de Wilt et al., 2016) verified continuous culture, which are displayed in Fig. 4, are larger than
that, under identical conditions, the removal of carbamazepine and those determined by Go mez et al. (2013) for a Muriellopsis sp.
trimethoprim was incomplete and did not exceed 30 and 60%, culture. In the present work, the JE determined for SaC was higher
respectively. Wang et al. (2016) studied the removal of phenol by than the values obtained for PC and the respective positive controls,
Chorella sp. culture, obtaining removal efficiencies up to 100% from either at dosage I or at dosage II. This evidences that the salicylic
an initial concentration of 500 mg l1 in seven days. Peng et al. acid treatment produced higher biomass by unit of radiation than
(2014) reported removals above 95% of progesterone by the control and the paracetamol treatment. Indeed, significantly
S. obliquus and Chlorella pyrenoidosa, nearly complete removal of larger (p 0.05) removal yields (JR) were determined under sal-
norgestrel by S. obliquus and almost 40% of norgestrel by icylic acid than under paracetamol either at dosage I (0.22 ± 0.01
C. pyrenoidosa. Likewise, Hom-Díaz et al. (2015) studied the elimi- gremoved E1 for SaCI >0.12 ± 0.01 gremoved E1 for PCI) or at dosage II
nation of the hormones E2 and EE2 from anaerobic digestate (2.45 ± 0.01 gremoved E1 for SaCII >1.72 ± 0.03 gremoved E1 for PCII).
78
Fig. 3. Average irradiance inside the culture of Chlorella sorokiniana for paracetamol (CþI , CþII -, PCI :, PCII A) (a) and salicylic acid (CþI , CþII -, SaCI :, SaCII A) (b) under
the initial pharmaceutical doses I (25 mg l1) and II (250 mg l1). Notes: Curves corresponding to CþI and CþII are represented both in (a) and (b) for comparison purpose. Ex-
periments were performed in triplicate and bars show standard derivations.
4. Conclusions than under paracetamol. The obtained results point to the prom-
issory application of C. sorokiniana as bioremediation system for the
An increased biomass productivity of C. sorokiniana occurred removal of paracetamol and salicylic from concentrated
under the presence of paracetamol or salicylic acid as compared wastewaters.
with the respective positive controls. Therefore, it may be
concluded that C. sorokininana was not only resistant to these Acknowledgments
pharmaceuticals at the considered dosages but also able to use
them as an additional source of organic carbon. During the batch Carla Escapa acknowledges the Spanish Ministry of Educations,
culture, larger maximum biomass concentration and carrying ca- Culture and Sports for her PhD fellowship (FPU12/03073). Sergio
pacity were attained under 250 mg l1 (dosage II) than 25 mg l1 Paniagua thanks the Spanish Ministry of Educations, Culture and
(dosage I) in the case of salicylic acid. Regarding pharmaceuticals Sports for his PhD fellowship (FPU14/05846). Also, Marta Otero
removal, in the batch culture, the maximum removal capacity was acknowledges support from the Spanish Ministry of Economy and
one order of magnitude higher under dosage II than I, both for Competitiveness, State Secretariat for Research, Development and
paracetamol and salicylic acid. Also, at the steady state of the Innovation (RYC-2010-05634).
semicontinuous culture, specific removal efficiencies were larger
under dosage II than under dosage I for both pharmaceuticals. In
Appendix A. Supplementary data
any case, higher removal yields were reached under salicylic acid
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.jenvman.2016.06.051.
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Deegan, A.M., Shaik, B., Nolan, K., Urell, K., Oelgemoeller, M., Tobin, J., Morrissey, A., to reduce pollution from the manufacturing of pharmaceuticals? Regul. Toxicol.
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Gattullo, C.E., Baehrs, H., Steinberg, C.E.W., Loffredo, E., 2012. Removal of bisphenol formation of progesterone and norgestrel by two freshwater microalgae (Sce-
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80
Supplementary material
Figure s1. Specific efficiency in the removal of pharmaceuticals during the batch culture for paracetamol (PCI , PCII ) (a) and
salicylic acid (SaCI , SaCII ) (b) under the initial pharmaceutical doses I (25 mg l-1) and II (250 mg l-1).
81
Figure s2. Specific efficiency in the removal of paracetamol and salicylic acid by Chlorella
sorokiniana at the steady state of the semicontinuous culture under the initial pharmaceutical
doses I (25 mg l-1) and II (250 mg l-1). Note: Experiments were performed in triplicate and bars
show standard derivations.
82
Table s1. Abbreviations used in the text for the controls and treatments with microalgae in the
set of experiments
Set of Set of
experiments experiments
Dosage I Dosage II
(25 mg l-1) (250 mg l-1)
Positive control C+I C+II
Treatment with
PCI PCII
microalgae
Paracetamol
Negative control CP-I CP-II
Treatment with
SaCI SaCII
microalgae
Salicylic acid
Negative control CSa-I CSa-II
83
84
Table s3 p-values from the Mann-Whitney test used for the comparison of treatments regarding
the kinetic parameters on growth and on the removal of pharmaceuticals during the batch
culture (n-1 degrees of freedom, n=3).
p- values for growth kinetic parameters

K a r
C+I-PCI 0.019 0.019 ≥ 0.999
C+I-SaCI 0.019 0.243 0.019
C+II-PCII 0.018 0.237 0.018
C+II-SaCII 0.018 0.018 0.018
PCI-PCII 0.019 0.019 0.559
SaCI-SaCII 0.019 0.243 0.019
PCI-SaCI ≥ 0.999 0.564 0.021
PCII-SaCII 0.018 0.018 ≥ 0.999
p- values for removal of pharmaceuticals kinetic parameters

K a r
PCI-PCII 0.019 ≥ 0.999 ≥ 0.999
SaCI-SaCII 0.019 0.019 0.243
PCI-SaCI 0.772 0.021 0.021
PCII-SaCII 0.018 0.018 0.018
the specific efficiency at the steady state of the semicontinuous culture (n-1 degrees of freedom,
n=3).
p- values
PCI-SaCI 0.050
PCII-SaCII 0.827
PCI-PCII 0.050
SaCI-SaCII 0.050
85
the quantum yield (ΨE) and removal yield (ΨR) at the steady state of the semicontinuous culture
(n-1 degrees of freedom, n=3).
p- values for ΨE p- values for ΨR
PCI-SaCI 0.513 PCI-SaCI 0.034
PCI-CI 0.513 PCII-SaCII 0.050
SaCI-CI 0.050
PCII-SaCII 0.050
PCII-CII 0.275
SaCII-CII 0.050
86
Capítulo 6
Artículo III
Comparison of the culture and harvesting of Chlorella vulgaris
and Tetradesmus obliquus for the removal of pharmaceuticals
from water
Journal of Applied Phycology, (2016) doi: 10.1007/s10811-016-1010-5
J Appl Phycol
DOI 10.1007/s10811-016-1010-5
Comparison of the culture and harvesting of Chlorella vulgaris

and Tetradesmus obliquus for the removal
of pharmaceuticals from water
C. Escapa 1 & R. N. Coimbra 1 & S. Paniagua 1 & A. I. García 1 & M. Otero 1
Received: 6 June 2016 / Revised and accepted: 6 November 2016

# Springer Science+Business Media Dordrecht 2016
Abstract The objective of this work was to assess and com- Keywords Algae . Emerging contaminants .
pare the removal efficiency of paracetamol and salicylic acid Phytoremediation . Bioremediation . Salicylic acid .
from aqueous medium by a microalgae-based treatment, using Paracetamol . Coagulation-flocculation
either Chlorella vulgaris or Tetradesmus obliquus. Moreover,
considering microalgae application in wastewater treatment,
the influence of these pharmaceuticals in the algal nutrient Introduction
removal capacity was evaluated. The removal of paracetamol
by T. obliquus (>40 %) was larger than by C. vulgaris (>21 %) Contaminants of emerging concern (CEC) include a wide
in batch culture, and this was also observed for salicylic acid range of compounds and may be defined as naturally occur-
(>93 % by T. obliquus and >25 % by C. vulgaris). Both strains ring, manufactured or manmade chemicals or materials that
removed nutrients (phosphate and nitrate) almost completely have now been discovered or are suspected to be present in
by the end of the batch culture, but T. obliquus showed the various environments and whose toxicity or persistence is
highest efficiency at the steady state conditions of the likely to alter the metabolism of a living organism significant-
semicontinuous culture. In spite of this, under the flocculants ly (Sauvé and Desrosiers 2014). Among them, pharmaceuti-
here tested, the efficiency in the recovery of biomass was cals have received special attention since diclofenac, 17-β-
much higher for C. vulgaris. These results highlight the im- estradiol (E2) and 17-α-ethynylestradiol (EE2) have been pro-
portance of strain selection in the application of microalgae for posed as priority hazardous substances within the EU Water
wastewater treatment and, particularly, for the removal of Framework Directive (Petrie et al. 2015).
pharmaceuticals. The presence of CECs in the environment is mainly attrib-
uted to the discharge of effluents from wastewater treatment
plants (WTP), which were not designed to remove these types
* M. Otero of compounds (Petrie et al. 2015). Thus, WTPs result in the
marta.otero@unileon.es discharge of CECs into receiving waters, these pollutants hav-
C. Escapa ing been detected at different concentrations in natural water
carla.escapa@unileon.es bodies (Ternes et al. 2004). In the specific case of pharmaceu-
R. N. Coimbra ticals, although acute toxicity has been often documented at
ricardo.decoimbra@unileon.es concentrations higher than those found in the aquatic environ-
ment, chronic toxicity can already occur at environmental
S. Paniagua
sergio.paniagua@unileon.es concentrations (Fent et al. 2006). Some of the reported effects
include the reduction of macroinvertebrate diversity in rivers
A. I. García
ana.garcia@unileon.es
(Muñoz et al. 2009), behavioural changes in mosquito fish
(Henry and Black 2008) and reproductive disruption in fish
1
Department of Applied Chemistry and Physics, Institute of
(Vajda et al. 2008). Therefore, given that legislation is expect-
Environment, Natural Resources and Biodiversity (IMARENABIO), ed to broaden to cover some of these CEC, it is a priority goal
University of León, 24071 León, Spain to improve their removal during wastewater treatment. In this
89
J Appl Phycol
sense and in view of sustainability, energy demand of waste- The inoculum of each strain was cultivated in 250-mL
water treatment must be reduced by the application of novel Erlenmeyer flasks in Mann and Myers medium (Mann and
treatment methods, with algae ponds having been proposed as Myers 1968) in a plant culture chamber, under controlled con-
a promising option for secondary effluent polishing (Petrie ditions: temperature (25 ± 1 °C), irradiance (175 μmol pho-
et al. 2015). tons m−2 s−1), photoperiod (12:12) and shaking (250 rpm).
Recently, phytoremediation of water by using photoauto- Bubbling column photobioreactors (PBRs) with spherical
trophic aquatic organisms such as algae has gained attention bases (40-mm diameter, 300-mm height, 300-mL capacity)
for the removal of CECs (Combarros et al. 2014; Hom-Díaz were used for the experimental set-up, keeping an operating
et al. 2015; de Wilt et al. 2016). Microalgae are characterized volume of 250 mL. In each PBR, the Mann and Myers culture
by high photosynthetic efficiency, high growth rates, wide medium was inoculated with the required volume of the cor-
adaptability and high potential to remove inorganic nutrients responding pre-cultured microalgae in order to have an initial
from the wastewater. The principal mechanism of algal nutri- concentration of about 3 × 106 cells mL−1.
ent removal is their uptake into the cell biomass. The main During the experimental phase, the culture was aerated
advantages of using microalgae for nutrient removal as a ter- with filtered air (0.22-μm sterile air-venting filter,
tiary treatment are the possibility of recycling the assimilated MillexFG50-Millipore), at a rate of 0.3 v/v min−1, enriched
nitrogen and phosphorus into algal biomass as a fertilizer, as a with CO2 at 7 % v/v, which was injected on demand to keep
source of products (e.g., paraffin, olefin, glycerol, protein, a constant pH (pH = 7.5 ± 0.5), as controlled by a pH sensor.
anti-oxidant, pigment, plastic, etc.), or as biofuel, and also The irradiance supplied during this phase was 370 μmol pho-
the generation of oxygenated high-quality effluent, as a result tons m−2 s−1, which was provided by eight fluorescent lamps
of photosynthesis (Matamoros et al. 2016). However, al- (58 W, 2150 lm, Philips, France). The photoperiod was main-
though the capability of microalgae wastewater treatment sys- tained at 12:12-h light/dark and the temperature at 25 ± 1 °C.
tems to remove organic matter and nutrients is well known
(Oswald et al. 1957; Oswald 1988), that is not the case of Removal of pharmaceuticals and nutrients
CECs. There is therefore the necessity for further studies on
the removal of these sorts of pollutants by algal systems Experimental set-up
(Petrie et al. 2015).
In this context, the aims of this study were (i) to determine PBRs were operated in batch mode until the end of the expo-
and compare the potential of green microalgae Chlorella nential growth phase, which was when the biomass concen-
vulgaris and Tetradesmus obliquus (previously named tration remained steady for two consecutive days. Then, the
Acutodesmus obliquus and Scenedesmus obliquus) to remove PBRs were operated under semicontinuous mode until the
paracetamol and salicylic acid from aqueous medium and (ii) growth parameters remained constant at the steady state.
to find out if the presence of these pharmaceuticals affects During the batch culture, an aliquot of 5 mL was taken daily
microalgal growth or nutrients removal capacity. from each PBR for the analytical determinations, this volume
Furthermore, considering that the separation of microalgae being replaced with distilled water to maintain the operating
from water is essential for the completion of treatment, differ- volume. During the semicontinuous culture, 30 % of the cul-
ent coagulants-flocculants at several dosages were tested for ture volume was harvested daily and used for analysis, this
each strain. The strains used in this work, namely Chlorella volume being replaced with fresh medium.
vulgaris and Tetradesmus obliquus, were selected since they For each strain (C. vulgaris and T. obliquus), two treat-
are known to have fast growth rates and potential for waste- ments were conducted: a treatment with inoculated culture
water treatment due to their tolerance to the severe environ- medium and 25 mg L−1 paracetamol (designated PCV with
mental conditions found in municipal wastewaters and some C. vulgaris and PTO with T. obliquus) and a treatment with
industrial wastewaters (Beuckels et al. 2015). inoculated culture medium and 25 mg L−1 salicylic acid (des-
ignated SCV with C. vulgaris and STO with T. obliquus). The
corresponding positive controls with inoculated culture medi-
um (CCV+ or CTO+, with C. vulgaris or T. obliquus, respec-
Materials and methods tively) were run. The negative controls consisted of
25 mg L−1 paracetamol (CP-) or salicylic acid (CS-) in culture
Microorganism and culture conditions medium with no microalgae. For each strain, experiments
were run in triplicate and under identical conditions in all the
The microalgae strains used in this study were Chlorella PBRs. Paracetamol (C8H9NO2, ≥99 %) was supplied by Roic
vulgaris SAG 221–12 and Tetradesmus obliquus SAG 276– Pharma and salicylic acid (C7H6O3, ≥99 %) by Panreac.
1, obtained from the SAG Culture Collection (University of Throughout the experiments, the growth of the culture was
Goettingen). daily monitored by the determination of biomass
90
J Appl Phycol
concentration and cell density. The removal of pharmaceuti- acid method, according to the Standard Methods 4500-P E
cals was daily determined by the analysis of the remaining (APHA 2008).
concentration of this drug in the culture medium. Moreover,
in order to assess the removal of nutrients, the concentrations Data analysis
of nitrate and phosphate were monitored daily. All analyses
were conducted in triplicate. Growth kinetics were resolved in OriginPro 8 using the classic
logistic model originally described by Verhulst (1838), which
is a good approximation to describe the growth of microalgae
Analytical methods (Xin et al. 2010). The logistic model fits a sigmoidal curve that
describes the relationship between microorganism growth and
Biomass concentration (Cb) was determined by optical density density in limited environmental conditions (Eq. 3).
at 680 nm (OD680) and verified by dry weight. Preliminary
K
studies were conducted to determinate the relationship be- N¼ ð3Þ
tween dry weight and OD680 for each strain, as shown in 1 þ ea−rt
Eq. (1) for C. vulgaris and in Eq. (2) for T. obliquus:
where N (g L−1) is the algal density at time t (h), K (g L−1) is
ODC:V 680 ¼ 2:7933 C b þ 0:0317; R2 ¼ 0:9958 ð1Þ the carrying capacity (the maximum algal density reached in
ODT :O 680 ¼ 2:0098 C b þ 0:0451; R2 ¼ 0:9915 ð2Þ the culture), a is a constant in the logistic model that refers to
the relative position from the origin and indicates the duration
Dry weight measurements were performed by filtering of the lag phase, and r (day−1) is the specific growth rate.
10 mL of culture through a 0.45-μm Whatman filter, which Furthermore, the kinetics of the removal of pharmaceuti-
was then washed with 20 mL HCl (0.5 M) to dissolve precip- cals and nutrients were fitted to the logistic model. In each
itated salts. Then the filtrate was dried in an oven at 80 °C for case, the parameter K (g L−1) is the maximum removal capac-
24 h. Additionally, the growth of the culture was measured as ity by the microalgae in the culture. The parameter a is a
cell density (Nc) by cell counting using a Neubauer chamber. constant in the logistic model that indicates the delay in the
The remaining pharmaceuticals concentration in the culture beginning of the target compounds removal, and the parame-
medium was quantified by a Waters HPLC 600 equipped with ter r (day−1) is the specific removal rate.
a 2487 Dual λ Absorbance Detector. A Phenomenex Gemini- Finally, differences among the strains with respect to the
NX C18 column (5 μm, 250 mm × 4.6 mm) was used for the kinetic parameters of growth, removal of pharmaceuticals and
separation. The wavelengths of detection were 246 and nutrients were compared by the Mann-Whitney U non-
236 nm for paracetamol and salicylic acid, respectively. The parametric test using IBM SPPS Statistics 21. Significance
mobile phase consisted of a mixture of acetonitrile/water was defined at p ≤ 0.05.
(30:70, v/v) for the analysis of paracetamol and a mixture of For the removal of pharmaceuticals and nutrients, the vol-
acetonitrile/water/orthophosphoric acid (70:30:0.1, v/v/v) for umetric efficiency for each target compound was calculated as
the analysis of salicylic acid. HPLC quality acetonitrile the difference between its average concentration in the influ-
(CH3CN) and orthophosphoric acid (H3PO4) from Prolabo ent (Cinf, mg L−1) and in the effluent (Cefflu, mg L−1) at every
Chemicals, and ultrapure water obtained by a Millipore sampling day, considering the daily dilution rate of the corre-
System were used for the preparation of the mobile phase. sponding operation stage (D, day−1) (Eq. (4)). During the
Before use, each mixture was passed through a Millipore batch culture, when a daily dilution rate was not performed,
0.45-μm pore size filter and degasified in an ultrasound bath these efficiencies are cumulatively expressed as mg L−1.
for 30 min. Before analysis, all the samples were centrifuged However, during the steady state of the semicontinuous cul-
twice at 5974×g for 10 min (SIGMA 2-16P centrifuge). For ture, these efficiencies are expressed as mg L−1 day−1, so to
the chromatographic analysis, the mobile phase flow rate was taking into account the daily dilution (0.3 day−1).
1 mL min−1 and the injection volume was 100 μL.
Nitrates (NO3−) were measured by the ultraviolet spectro- Volumetric e f f iciency mg L−1 day−1 ¼ C in f −C e f f lu D ð4Þ
photometric screening method, according to Standard
Methods 4500-NO3− B (APHA 2008). In the treatments with The volumetric efficiency, at the end of the batch culture
paracetamol, and to avoid absorbance-related interferences at and during the steady state of the semicontinuous culture, was
the wavelength of detection, nitrates were measured by the also expressed as percentage for an easier understanding of the
visible spectrophotometric method described by Doane and results (Eq. (5)).
Horwath (2003). This method reduces nitrate to nitrite using
vanadium (III) in acid solution, performing the measurement C inf −C efflu
Volumetric efficiency ð%Þ ¼ 100 ð5Þ
at 540 nm. Phosphates (PO43−) were measured by the ascorbic C inf
91
J Appl Phycol
The specific efficiency of the removed pharmaceuticals and All experiments were run in triplicate, under room temper-
nutrients was calculated as the ratio between the volumetric ature (25 ± 1 °C) and at the pH of the culture medium
efficiency and the biomass concentration (Cb, g L−1) (Eq. (6)). (7.5 ± 0.5).
Likewise, during the batch culture, these efficiencies are cu-
mulatively expressed as mg removed g biomass −1 and as Analytical methods
mgremoved gbiomass−1 day−1 during the steady state of the
semicontinuous culture. Biomass concentration was measured by optical density at
−1
680 nm. Biomass recovery was calculated from the biomass
Speci f ic e f f iciency mg removed g−1
biomass day
concentration ratio at the clarified zone (Cbt) versus the cul-

C in f −C e f f lu D ture concentration at the beginning of the experiment (Cbi),
¼ ð6Þ Eq. (7).
Cb

Cbt
Recovery of biomass ¼ 1− 100 ð7Þ
Cbi
Recovery of microalgae biomass
by coagulation-flocculation
Experimental set-up Results
Metal salts, synthetic polyelectrolytes and a biopolymer were Removal of pharmaceuticals and nutrients
tested to determine the minimum dosage that ensures recovery
efficiencies of biomass higher than 95 %. Iron chloride Growth of the culture
(FeCl3) and aluminium chloride (AlCl3) were used as metal
salts, supplied by Panreac. Synthetic polyelectrolytes were The microalgae growth curves of C. vulgaris and T. obliquus in
supplied by Chemipol, sold as chemifloc CH-35 (strong cat- the presence of paracetamol, the corresponding positive controls
ionic polymers) and chemifloc CH-15 (medium cationic poly- and their respective fittings to the logistic kinetic model are rep-
mers), both with high molecular weight and used in industrial resented as values of biomass concentration versus time in
and urban wastewater treatment processes. Chitosan was sup- Fig. 1a. Differences among treatments were analysed according
plied by Acofarma as biopolymer. Doses of each coagulant to growth kinetic parameters, whose results are shown in Table 1.
were expressed as milligram of coagulant per gram of The values obtained for the parameter a in Table 1 indicate
microalgal biomass. that the addition of paracetamol produced a significantly de-
A first series of experiments was performed to study the layed the response in beginning of the exponential growth
efficiency of five doses of each coagulant with the biomass phase for C. vulgaris compared with the positive control.
harvested at the end of the batch culture. The doses tested were However, the lag phase of T. obliquus was not significantly
100, 150, 200, 250 and 300 mg g−1 for metal salts, 2.5, 5, 10, modified by the presence of paracetamol. The carrying capac-
15 and 20 mg g−1 for synthetic polyelectrolytes and 20, 40, ity was increased above 31 % in the presence of paracetamol
100, 150, 200 mg g−1 for chitosan. Then, on the basis of the in the C. vulgaris culture compared with the positive control.
results from this first stage, a second series of trials was carried However, T. obliquus culture was not significantly modified
out at the semicontinuous mode to verify the efficiencies of by the addition of the drug, and the maximum algal density
the best dose of each coagulant. reached in the treatment was similar to the positive control. In
The biomass used for the coagulation-flocculation experiments spite of the difference of the strains to the presence of para-
was obtained from the PBRs used for the culture of C. vulgaris cetamol, there were no significant differences between para-
and T. obliquus, which, as above described, were run in batch cetamol treatments, even though the value reached for the
mode until the exponential growth finished, followed by parameter K in the case of T. obliquus positive control was
semicontinuous mode with a 30 % dilution rate of culture volume. 24 % greater than for the C. vulgaris-positive control. On the
Flocculation assays were performed following the same other hand, the specific growth rate (r) was 1.3 times higher in
methodology described in Escapa et al. (2015). The suspension the paracetamol treatment for C. vulgaris than in the positive
was placed in the test tube, in which the flocculation efficiency control. However, likewise the K parameter, the specific
was determined after 0, 5, 10, 15, 30 and 60 min. At those times, growth rate was neither modified in the case of T. obliquus
samples of 1 mL were taken from the clarified zone to measure treatment compared with the corresponding positive control.
the corresponding biomass concentration. As positive control, In addition, when comparing the two strains, there were no
the microalgae culture with no flocculant was placed in the test significant differences between the paracetamol treatments
tube and flocculation efficiency was measured over 24 h. despite significant differences between the positive controls.
92
J Appl Phycol
Fig. 1 Growth curves of

C. vulgaris (CCV+, PCV) and
T. obliquus (CTO+, PTO) for the
paracetamol treatments (a) and
growth curves of C. vulgaris
(CCV+, SCV) and T. obliquus
(CTO+, STO) for the salicylic
acid treatments (b). The
continuous lines correspond to the
fitted logistic kinetic model
during batch culture. Experiments
were performed in triplicate and
bars show standard derivations.
Note: experimental data points
obtained during semicontinuous
culture are connected with dashed
lines
Figure 1b depicts the microalgal biomass growth curves un- the specific growth rate of STO was significantly lower than
der the presence of salicylic acid, the corresponding positive that of SCV, and the lowest among all the treatments.
controls and their respective fitted logistic kinetic model as During the semicontinuous mode, daily dilutions rates pro-
values of biomass concentration versus time. Moreover, differ- duced instability, as for adaptation of the microalgae culture
ences among treatments were analysed according to growth when the growth conditions change. Consequently, the
kinetic parameters, whose results are shown in Table 1. growth rate declined with respect to the batch mode, and then,
The a values in Table 1 show that the addition of salicylic the growth parameters remained stable at the steady state.
acid in C. vulgaris produced the longest lag phase among all In view of the obtained results, it may be concluded that,
the treatments. Moreover, as compared with their respective under the here considered drugs, the culture of C. vulgaris
positive control, the increase of the C. vulgaris lag phase was showed an increased lag phase, maximum biomass produced
greater in the presence of salicylic acid compared to paracet- (>31 % PCV, >18 % SCV) and growth rate (more than 1.3 times
amol. In the case of T. obliquus, a significant larger a value for PCV and 2.0 times for SCV) as compared with positive
was obtained in the positive control than with salicylic acid. controls. In addition, the effects on the lag phase and growth
Comparing the two treatments with salicylic acid, C. vulgaris rate were more pronounced in the presence of salicylic acid than
showed a quite longer lag phase than T. obliquus. paracetamol, in spite of the biomass concentration at the end of
For both strains, the maximum algal density reached in the the batch culture being higher under paracetamol than salicylic
culture in the presence of salicylic acid was significantly higher acid. In the case of T. obliquus, the addition of paracetamol did
than in their respective positive controls. Moreover, under not modify microalgae kinetic parameters with respect to the
salicylic acid, the carrying capacity of T. obliquus was 34 % positive controls. However, under the addition of salicylic acid,
greater than that of C. vulgaris. In spite of this, the maximum the culture of T. obliquus showed a shorter lag phase, an in-
biomass reached by the C. vulgaris culture did not show signif- creased K value (>36 %), but a slower growth rate (<0.6 times
icant differences under paracetamol and salicylic acid. lower) than the corresponding positive control.
Differently, the maximum biomass of T. obliquus was larger
under salicylic acid since the parameter K obtained with this drug Removal of pharmaceuticals
was significantly higher than under paracetamol and was the
highest among all the treatments. The concentrations of both pharmaceuticals decreased over the
The C. vulgaris specific growth rate was 2.0 times higher in time in the treatments with microalgae, either with C.vulgaris or
the presence of salicylic acid than in the positive control, but it T. obliquus. Meanwhile, no reduction by photodegradation and/
was significantly reduced in the case of T. obliquus. Moreover, or volatilization was observed in the negative controls.
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J Appl Phycol
Table 1 Experimental data (Cbo, Nco, Cbm, Ncm) and logistic model kinetic parameters (K, a, r) determined for the growth of positive controls and treatments with paracetamol and salicylic acid of
6.97 × 107 ± 2.02 × 106
Cbo initial biomass, Nco initial number of cells, Cbm maximum biomass, Ncm maximum number of cells, K carrying capacity, a constant of logistic kinetic model, r specific microalgae growth rate, R2
Therefore, it may be assumed that the pharmaceutical concen-
tration decrease in the microalgae treatments was due to removal
by the microalgae.
4.33 ± 0.30
4.71 ± 0.30
0.72 ± 0.01
4.20 ± 0.09
The removal curves of pharmaceuticals by each strain of
8.35 × 105
microalgae and the corresponding fitted logistic kinetic model
0.9883
STO
0.08 during the batch mode are displayed in Fig. 2a for paracetamol
treatments and Fig. 2b for salicylic acid treatments. In addi-
tion, the results from the fitted models are displayed in
4.62 × 107 ± 1.95 × 106 Table 2. For both microalgae strains, these removal curves
displayed similar sigmoidal trend than growth curves
(Fig. 1a, b), which indicates a relationship between microalgal
3.09 ± 0.20
3.27 ± 0.15
1.12 ± 0.03
4.97 ± 0.31
growth and the removal of pharmaceuticals.

8.35 × 105
0.9886 Regarding the parameter a, there were no significant dif-

PTO
0.08
ferences between C. vulgaris and T. obliquus in the beginning

of the removal of either paracetamol or salicylic acid.
Moreover, C. vulgaris displayed similar values for the param-
4.77 × 107 ± 1.41 × 105
eter a under the presence of both pharmaceuticals. On the

contrary, T. obliquus showed a delayed response in the begin-
ning of the removal of salicylic acid as compared with
paracetamol.
3.33 ± 0.06
3.46 ± 0.08
1.16 ± 0.07
5.45 ± 0.43
8.35 × 105
The parameter K obtained for paracetamol revealed that

0.9874
CTO+
0.08
T. obliquus reached a carrying capacity 1.7 times higher than

C. vulgaris. The same was observed in the salicylic acid treat-
ments, T. obliquus displaying a maximum removal capacity
1.76 × 108 ± 4.90 × 107
3.8 times higher than C. vulgaris. On the other hand,

C. vulgaris reached similar maximum removal capacities for
both pharmaceuticals, while the maximum removal capacity
of salicylic acid by T. obliquus was 2.4 times higher than that
3.02 ± 0.27
7.99 ± 0.41
3.09 ± 0.23
1.69 ± 0.13
1.21 × 106
of paracetamol. The specific removal rates revealed no signif-

0.9929
SCV
0.11
icant differences among the treatments.

As a consequence of the different responses obtained for
the removal parameters between both strains, at the end of the
2.17 × 108 ± 1.97 × 107
batch culture, efficiencies above 21 % for PCV, 25 % for SCV,

40 % for PTO and 93 % for STO were achieved. These results
indicated a greater removal capacity of both pharmaceuticals
C. vulgaris (CCV+, PCV, SCV) and T. obliquus (CTO+, PTO, STO)
by T. obliquus than by C. vulgaris.

3.35 ± 0.08
5.58 ± 0.03
3.42 ± 0.15
1.08 ± 0.04
1.21 × 106
The average volumetric efficiencies on the removal of

0.9968
PCV
pharmaceuticals by each strain at the steady stage of the

0.11
semicontinuous culture are shown in Fig. 2. The paracetamol

volumetric efficiency did not show large differences between
1.18 × 108 ± 8.84 × 106
the strains, reaching values above 12 % for C. vulgaris and

Error bars represent standard deviation (n = 3)
9 % for T. obliquus. However, the salicylic acid volumetric

efficiency of T. obliquus (above 99 %) was 4.3 times greater
than that of C. vulgaris (22.91 %). Moreover, the ratios be-
4.47 ± 0.25
2.60 ± 0.15
0.84 ± 0.06
2.48 ± 0.11
1.21 × 106
tween the volumetric efficiency and the microalgal biomass

0.9971
CCV+
are shown in Table 2 as specific efficiencies. The obtained

0.11
specific efficiencies revealed that C. vulgaris cells removed

correlation coefficient
greater than 2.1 times more salicylic acid than paracetamol,

Ncm (cells mL−1)
and T. obliquus removed 5.0 times more salicylic acid than

Nc0 (cells mL−1)
paracetamol, per gramme of biomass. On the other hand, the

Cbm (g L−1)
Cb0 (g L−1)
paracetamol removal per gram of biomass was similar for both

K (g L−1)
r (day−1)
strains, while the salicylic acid removal by T. obliquus was 2.8

R2
times greater than by C. vulgaris.

a
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J Appl Phycol
Fig. 2 Volumetric efficiency in the removal of paracetamol by batch culture. Volumetric efficiency in the removal of paracetamol (c) and
C. vulgaris (PCV) and T. obliquus (PTO) during batch culture (a) and salicylic acid (d) at the steady state of the semicontinuous culture.
in the removal of salicylic acid by C. vulgaris (SCV) and T. obliquus Experiments were performed in triplicate and bars show standard devia-
(STO) during batch culture (b). Dots correspond to experimental data tions. Note: the scale of the y-axis in Fig. 2c and Fig. 2d is different and
and continuous lines correspond to the fitted logistic kinetic model during has been adjusted for the appropriate visualization of results
In view of the obtained results, it may be concluded that the culture are shown in Fig. 3. Like for the pharmaceuticals,
salicylic acid was more efficiently removed than paracetamol these removal curves have a similar sigmoidal trend as the
by both strains. In addition, for both salicylic acid and para- growth curves (Fig. 1) since microalgal growth is given by
cetamol, T. obliquus displayed higher removal capacity, either the nutrient uptake.
per litre or per gramme of biomass, than C. vulgaris. Results from the fits of the removal of nutrients to the
logistic model are displayed in Table 3. Regarding the remov-
Removal of nutrients al of nitrates, the parameter a revealed that the addition of
paracetamol and salicylic acid did not change the lag phase
The removal curves of nitrates and phosphates by each strain of C. vulgaris as compared with the positive control.
of microalgae and their fitted logistic models during the batch However, in the T. obliquus culture, the presence of these
Table 2 Logistic model kinetic

parameters (K, a, r) determined PCV PTO SCV STO
for the removal of paracetamol
and salicylic acid in the batch a 3.84 ± 0.01 3.19 ± 0.58 4.09 ± 0.87 4.11 ± 0.16
culture of C. vulgaris (PCV, SCV) K (mg L−1) 6.23 ± 0.02 10.41 ± 1.58 6.44 ± 0.63 24.67 ± 0.32
and T. obliquus (PTO, STO) r (day−1) 0.77 ± 0.01 0.86 ± 0.21 0.84 ± 0.17 0.76 ± 0.03
R2 0.9827 0.9766 0.9947 0.9973
Volumetric efficiency (mg L−1 day−1) 0.95 ± 0.05 0.72 ± 0.07 1.72 ± 0.15 7.55 ± 0.01
Specific efficiency (mg g−1 biomass day−1) 0.32 ± 0.02 0.37 ± 0.03 0.67 ± 0.06 1.85 ± 0.02
Volumetric efficiency and specific efficiency attained in the steady state of the semicontinuous culture. Error bars
represent standard deviation (n = 3)
K carrying capacity, a constant of logistic kinetic model, r specific pharmaceutical removal rate, R2 correlation
coefficient
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J Appl Phycol
Fig. 3 Volumetric efficiency in the removal of nitrates (a) and culture. Dots correspond to experimental data and continuous lines
phosphates (b) by C. vulgaris (CCV+, PCV) and T. obliquus (CTO+, correspond to the fitted logistic kinetic model during batch culture.
PTO) in the paracetamol treatments. Volumetric efficiency in the Experiments were performed in triplicate and bars show standard
removal of nitrates (c) and phosphates (d) by C. vulgaris (CCV+, SCV) deviations
and T. obliquus (CTO+, STO) in the salicylic acid treatments during batch
Table 3 Logistic model kinetic parameters (K, a, r) determined for the removal of nitrates and phosphates in the batch culture of C. vulgaris treatments
(CCV+, PCV, SCV) and T. obliquus treatments (CTO+, PTO, STO)
CCV+ PCV SCV CTO+ PTO STO
Nitrates a 4.25 ± 0.38 3.84 ± 0.27 3.95 ± 0.66 3.72 ± 0.39 5.07 ± 1.09 4.73 ± 0.49
K (mg L−1) 653.62 749.09 719.01 671.97 700.85 732.48
± 23.89 ± 28.59 ± 1.75 ± 25.71 ± 19.19 ± 18.00
r (day−1) 0.78 ± 0.07 0.71 ± 0.11 0.88 ± 0.16 0.94 ± 0.14 1.11 ± 0.23 0.95 ± 0.09
R2 0.9899 0.9826 0.9736 0.9894 0.9791 0.9871
Volumetric efficiency (mg L−1 day−1) 175.08 ± 1.50 178.82 ± 1.65 204.79 214.85 ± 0.44 206.97 ± 1.57 217.84 ± 0.35
± 3.09
Specific efficiency 79.30 ± 1.44 62.11 ± 1.07 79.11 ± 1.69 120.79 ± 4.01 101.61 ± 1.17 53.38 ± 0.63
(mg g−1 biomass day−1)
Phosphates a 6.33 ± 0.77 6.52 ± 0.65 5.40 ± 0.99 2.86 ± 0.26 2.55 ± 0.43 3.59 ± 0.45
K (mg L−1) 53.85 ± 1.01 54.34 ± 0.14 54.88 ± 0.51 53.38 ± 0.36 53.67 ± 0.42 54.41 ± 0.83
r (day−1) 1.25 ± 0.13 1.51 ± 0.16 1.42 ± 0.26 2.72 ± 0.21 2.29 ± 0.60 1.99 ± 0.24
R2 0.9928 0.9951 0.9966 0.9863 0.9823 0.9905
Volumetric efficiency (mg L−1 day−1) 15.81 ± 0.03 16.07 ± 0.12 16.16 ± 0.06 16.29 ± 0.05 16.28 ± 0.01 16.31 ± 0.01
Specific efficiency 6.94 ± 0.10 5.17 ± 0.09 6.69 ± 0.73 9.08 ± 0.23 8.01 ± 0.14 4.00 ± 0.05
(mg g−1 biomass day−1)
Volumetric efficiency and specific efficiency attained in the steady state of the semicontinuous culture. Error bars represent standard deviation (n = 3)
K carrying capacity, a constant of logistic kinetic model, r specific removal rate, R2 correlation coefficient
96
J Appl Phycol
pharmaceuticals significantly increased the lag phase. The After batch culture, during the steady state of the
maximum removal capacity of nitrates was improved above semicontinuous culture, as shown in Table 3, the volumetric
14 % by the addition of paracetamol in the C. vulgaris culture, efficiency of nitrate removal in the C. vulgaris culture was 1.2
but it remained the same in the case of T. obliquus, as com- times lower than the removal of phosphate, which was nearly
pared with their respective positive controls. The presence of complete (Fig. 4). Meanwhile, in the T. obliquus culture, the
salicylic acid improved significantly the nitrate removal ca- removal of both nutrients was nearly complete at the steady
pacity of both strains (>10 % SCV, >9 % STO) as compared state. The specific efficiencies on the removal of nutrients
with their respective positive controls. The values of nitrate (Table 3) by T. obliquus in the positive control and in the
removal capacity by both strains under the considered phar- presence of paracetamol were markedly larger than by
maceuticals were very similar. Finally, the results obtained for C. vulgaris-positive control and paracetamol treatment, re-
the nitrate-specific removal rates revealed no significant dif- spectively. However, under salicylic acid, C. vulgaris
ferences among the treatments, all achieving efficiencies of displayed higher specific efficiencies than T. obliquus, due
82–97 % at the end of the batch culture. to the larger biomass of the latter.
As regards the removal of phosphates, according to the In view of the above, it may be highlighted that T. obliquus
parameter a, the addition of pharmaceuticals did not modify displayed a faster nutrient removal kinetics, despite both
the lag phase of either strain, as compared with the corre- strains being able to completely remove both nutrients by
sponding positive control. However, as indicated by a values the end of the batch culture and at the steady state of the
(Table 3) and Fig. 3, the response in the removal of phosphates semicontinuous culture.
by C. vulgaris was significantly longer than by T. obliquus
under both pharmaceuticals. The maximum removal capacity Recovery of microalgae biomass
of phosphates was the same for all treatments and positive by coagulation-flocculation
controls, with no significant differences between strains or
drugs. On the other hand, although the presence of paraceta- The coagulation-flocculation assays were performed with the
mol did not affect the specific removal rate of phosphates by microalgae biomass recovered at the end of the batch culture.
C. vulgaris, it was reduced in the case of T. obliquus, as com- Natural sedimentation of C. vulgaris was practically non-ex-
pared with their respective positive controls. This reduction istent, even with incubation times greater than 60 min (results
was also verified to occur for both strains under the addition of not shown), whereas the flocculation ability of T. obliquus (5–
salicylic acid. However, when comparing both strains, 10 μm) was greater, reaching recovery efficiencies above
T. obliquus showed a quicker phosphate-specific removal 26 % in 60 min and complete recovery after 16 h.
rates than C. vulgaris both in the presence of paracetamol The flocculation results for C. vulgaris are shown in
and salicylic acid. Despite the differences among these specif- Fig. 5. Between metal salts, it can be observed that
ic removal rates, at the end of the batch culture, efficiencies AlCl3 (Fig. 5a), which at the lowest doses (100 and
above 95 % were achieved in all the treatments. In any case, at 150 mg g -1 ) provided higher efficiencies than FeCl 3
the end of the batch culture, the removal of both nutrients by (Fig. 5b), did not show great differences among the dos-
the strains here considered was nearly complete since the end ages tested. However, at the highest doses (200, 250 and
of the exponential growth phase was caused by the depletion 300 mg g −1 ), FeCl 3 reached higher efficiencies in a
of nutrients. slightly shorter time than AlCl3. The doses that proved
Fig. 4 Volumetric efficiencies of

C. vulgaris and T. obliquus in the
removal of nitrates (solid bars)
and phosphates (raster bars) at
the steady state of the
semicontinuous culture.
Experiments were performed in
triplicate and bars show standard
deviations
97
J Appl Phycol
Fig. 5 Recovery efficiency of Chlorella vulgaris biomass at the end of a coagulant per gram of biomass. Experiments were performed in triplicate
batch culture with different doses of AlCl3 (a), FeCl3 (b), CH-35 (c), CH- and bars show standard deviations
15 (d), and chitosan (e). The doses are expressed as milligram of
to be more efficient were 250 mg g − 1 for AlCl 3 needed to reach T. obliquus recoveries of 88.43 ± 0.21 % using
(95.16 ± 1.20 %, 5 min) and 200 mg g−1 for FeCl3 AlCl3 and 98.95 ± 0.88 % with FeCl3. However, using these
(95.01 ± 2.55 %, 1 min). Furthermore, incubation times salts, incubation times higher than 15 min led to an improve-
longer than 15 min led to a recovery improvement of ment of the average recovery of up to 35 % for AlCl3 and
less than 5 %, except for the lowest doses of FeCl3 20 % for FeCl3. These further recoveries after 15 min may
(100 and 150 mg g−1), which showed an improvement be explained by the effect of natural flocculation. Still, in the
up to 17 %. case of FeCl3, cell colour changes were observed under a high
Flocculation results with metal salts for T. obliquus are concentration doses (200, 250 and 300 mg L−1), the superna-
shown in Fig. 6a and b, for AlCl3 and FeCl3, respectively. tant becoming yellowish, as also shown by Papazi et al.
The efficiencies reached by AlCl3 were clearly poorer than (2010).
those obtained by FeCl3, both salts giving poorer recoveries Results of flocculation of C. vulgaris by synthetic polyelec-
for T. obliquus than for C. vulgaris. In fact , under the highest trolytes CH-35 and CH-15 are shown in Fig. 5c and d, respec-
dose tested (300 mg L−1), incubation times of 60 min were tively. Both flocculants gave similar results with efficiencies
98
J Appl Phycol
Fig. 6 Recovery efficiency of Tetradesmus obliquus biomass at the end coagulant per gram of biomass. Experiments were performed in triplicate
of a batch culture with different doses of AlCl3 (a), FeCl3 (b), CH-35 (c), and bars show standard deviations
CH-15 (d), and chitosan (e). The doses are expressed as milligramm of
higher than 99.5 % achieved with 10 mg g−1 of CH-35 or CH- incubation for 15 min. Incubation times of 60 min were nec-
15 (1-min incubation time). As for the metal salts, incubation essary to reach T. obliquus recoveries of 74.49 ± 2.09 % with
times longer than 15 min did not meant an improvement of CH-35 and 70.11 ± 3.78 % with CH-15, both at the highest
biomass recovery, except for the lowest dosage (under 2.5 mg dose tested (20 mg L−1). Differently from C. vulgaris, incu-
g-1 of either CH-35 or CH-15, biomass recovery increased bation times longer than 15 min led to an improvement in
about 10% from 15 to 60 min incubation time). recovery of up to 17 % with CH-35 and up to 23 % with
The results of flocculation of T. obliquus with CH-35 and CH-15.
CH-15 are shown in Fig. 6c and d, respectively. The strong The quick efficiency and the low improvement of biomass
cationic polymer (CH-35) performed slightly better than the recovery for times longer than 15 min with C. vulgaris bio-
medium cationic polymer (CH-15), although both provided mass may be explained by the fact that a single big floc was
poorer recoveries for T. obliquus than for C. vulgaris. generated immediately after the addition of the coagulant in
Likewise, none of the doses tested reached efficiencies higher the cases where high efficiencies were obtained. However, in
than 55 % for CH-35 and 49 % for CH-15, even with the case of T. obliquus, increasing incubation times resulted in
99
J Appl Phycol
larger recoveries, since the efficiencies achieved in the first higher (1.68 day−1 in both cases) than those here determined.
minutes were due to the coagulant addition and subsequently However, these relatively high growth rates determined by
to the natural flocculation ability of the cells. Given the rela- Zhang et al. (2012a) were reduced to 1.2 day−1 by the addition
tive low dose of synthetic polyelectrolytes here used, these are of 10 mg L−1 of carbamazepine. In the present study, the
comparatively quicker and more efficient flocculants. addition of paracetamol meant an increase of the growth rate
Flocculation results obtained with chitosan are shown in as compared with the positive control in both strains.
Fig. 5e for C. vulgaris and Fig. 6e for T. obliquus. For the Meanwhile, the addition of salicylic acid led to an increase
tested doses, the recoveries provided by chitosan were as low of the C. vulgaris growth rate, but decreased the growth rate of
as around 53 % for C. vulgaris and 36 % for T. obliquus (60- T. obliquus culture.
min incubation time). In a previous study with C. sorokiniana under identical
The utilization of the above flocculating agents (FeCl3, culture conditions and drug dosage (Escapa et al. 2015), the
AlCl3, CH-35, CH-15 and chitosan) for the recovery of the specific growth rate in the presence of paracetamol
biomass harvested in the steady state (data not shown) provid- (0.96 day−1) was close to the rates obtained by C. vulgaris
ed lower efficiency values than those achieved with the same (1.08 day−1) and by T. obliquus (1.12 day−1) in the present
dosage in the batch stage (Figs. 5 and 6). In the case of AlCl3, study. As compared with paracetamol, under the addition of
maximum recoveries of 83.31 ± 1.71 and 76.85 ± 1.22 % were the same dose of salicylic acid, Escapa et al. (2015) deter-
achieved in 60-min incubation time for C. vulgaris (with mined lower specific growth rates (0.77 day −1 ) for
250 mg g−1 AlCl3) and T. obliquus (with 300 mg g−1 AlCl3), C. sorokiniana. This was also observed for T. obliquus in this
r e s p e c t iv e l y. Fo r F e C l 3 , biomass recov ered w as work, with similar specific growth rates (0.72 day−1). On the
87.26 ± 1.47 % of C. vulgaris (with 200 mg g−1 FeCl3) and contrary, comparative larger specific growth rates were here
83.82 ± 2.07 % of T. obliquus (with 300 mg g−1 FeCl3) after determined for C. vulgaris (1.69 day−1) in the presence of
60-min incubation. In the case of the synthetic polyelectro- salicylic acid. However, as compared with positive controls,
lytes, the efficiencies were reduced up to 73.63 ± 2.18 and the addition of paracetamol in the C. sorokiniana culture
67.35 ± 1.81 % in the case of C. vulgaris culture when using (Escapa et al. 2015) increased the maximum algal density
CH-35 or CH-15, respectively (10 mg g−1, 1-min incubation (Cbm) above to 49 % and the addition of salicylic acid above
time), and to 57.45 ± 0.84 and 52.33 ± 1.93 % for T. obliquus 52 %, which are larger increases than those obtained in the
with CH-35 and CH-15, respectively (20 mg g−1, 60-min in- present study for C. vulgaris and T. obliquus. On the other
cubation time). Finally, the recovery efficiencies achieved by hand, the lag phase of C. sorokiniana (Escapa et al. 2015)
chitosan were 39.22 ± 3.03 % for C. vulgaris and of was longer under pharmaceutical culture, which is coincident
29.75 ± 2.56 % for T. obliquus (with 200 mg g−1 chitosan, with the here observed for C. vulgaris. Therefore, on the
60-min incubation time). whole, the obtained results point to an increase in biomass
growth in the presence of the studied pharmaceuticals, which
mean an additional carbon source (Escapa et al. 2015). This
Discussion increase was similar for both strains of Chlorella, while
C. sorokiniana culture displayed a more remarkable increase
Growth rates attained in this research by C. vulgaris (CCV+ of biomass concentration in the presence of the studied drugs.
0.84 ± 0.06, PCV 1.08 ± 0.04, SCV 1.69 ± 0.13 day−1) and Regarding the removal of pharmaceuticals, it should be
T. obliquus (CTO+ 1.16 ± 0.07, PTO 1.12 ± 0.03, STO noted that in a previous study under identical operation con-
0.72 ± 0.01 day−1) was mostly higher than those reported by ditions (Escapa et al. 2015), C. sorokiniana displayed efficien-
other authors in wastewater. Wang et al. (2016b) reported cies above 67 % for paracetamol and 73 % for salicylic acid at
specific growth rates for C. vulgaris and A. obliquus, cultivat- the end of the batch culture. Then, at the steady state of the
ed under different photoperiods in sterile domestic effluent, all semicontinuous culture, C. sorokiniana showed efficiencies
of them lower than 0.4 day−1. Zhao et al. (2015) also described above 41 % for paracetamol and 93 % for salicylic acid.
specific growth rates 0.36 day−1 for C. vulgaris and 0.45 day−1 Therefore, among these strains, C. sorokiniana and
for S. obliquus cultivated in sterile domestic effluent. T. obliquus showed the highest removal capacity for paracet-
Domínguez Cabanelas et al. (2013) described specific growth amol and salicylic acid, respectively. Meanwhile, C. vulgaris
rates for C. vulgaris close to 0.38 day−1 in different domestic showed an intermediate removal capacity for each
wastewaters. On the other hand, similar to the here determined pharmaceutical.
specific growth rates were those reported by Arbib et al. Published results on the removal of emerging contaminants
(2013) for S. obliquus cultivated in urban wastewater, which by microalgae have revealed different efficiencies depending
were about 1 day−1. Furthermore, in HB-4 artificial medium, on the pollutant and on the microalgal strain. For example,
growth rates attained by S. obliquus and Chlorella Gattullo et al. (2012) demonstrated that Monoraphidium
pyrenoidosa after 144-h incubation (Zhang et al. 2012a) were braunii was able to remove up to 48 % of bisphenol A with
100
J Appl Phycol
an initial concentration of 4 mg L−1. De Wilt et al. (2016) here obtained for C. vulgaris and much better than for
reported removal efficiencies by C. sorokiniana, grown in T. obliquus. At the end of the batch culture of
wastewater streams, of up to 60–100 % for diclofenac, ibupro- Scenedesmus sp., Selesu et al. (2016) reported biomass
fen, paracetamol and metoprolol. However, under identical recoveries of 96.5 ± 1.1 % using 300 mg L−1 of Al2
conditions, the removal of carbamazepine and trimethoprim (SO4)3 and 96.0 ± 0.8 % using 150 mg L−1 of FeCl3, both
was incomplete and did not exceed 30 and 60 %, respectively with incubation times of 30 min. Considering biomass
(de Wilt et al. 2016). Wang et al. (2016a) studied the removal of concentration, the recoveries obtained by Selesu et al.
phenol by Chorella sp. culture, obtaining removal efficiencies (2016) with the ferric salt gave slightly better results than
up to 100 % from an initial concentration of 500 mg L−1 in the results obtained in this work, whereas the contrary
7 days. Peng et al. (2014) reported removals above 95 % of occurred with the aluminium salt. These efficiencies are
progesterone by S. obliquus and C. pyrenoidosa, nearly com- much lower than those here reported due to the high doses
plete removal of norgestrel by S. obliquus and almost 40 % used and long incubation times.
removal of norgestrel by C. pyrenoidosa. Likewise, Hom- The use of cationic polymers has been previously re-
Díaz et al. (2015) studied the elimination of the hormones E2 ported (Granados et al. 2012) for microalgae flocculation
and EE2 from an anaerobic digestate centrate by the microalgae although, the optimum doses depend on the nature of the
Selenastrum capricornutum and Chlamydomonas reinhardtii. polymer. However, a main disadvantage of these poly-
After 7 days of culture, they determined removals above 88 % electrolytes use is floc viscosity, which caused adhesion
for E2 and above 60 % for EE2. of cell aggregates to the wall of the test tubes. From a
The obtained results with respect to the removal of nitrates practical point of view, this may mean an important lim-
and phosphates confirm the feasibility of using the C. vulgaris itation for the retrieval and subsequent use of the so re-
and T. obliquus for the removal of nutrients and their applica- covered microalgal biomass.
bility in wastewater treatments, as has been previously report- The results obtained with chitosan are in agreement with
ed by others (Arbib et al. 2013; Cabanelas et al. 2013; Gómez- the low recoveries (around 15 %) achieved in previous assays
Serrano et al. 2015; Prandini et al. 2016). However, it is nec- with C. sorokiniana (Escapa et al. 2015) and with results re-
essary to highlight the existence of a nutrient limitation in ported by other researchers (Granados et al. 2012).
WTPs, with a typical concentration of ammonia nitrogen of Lower recoveries of biomass at the steady sate of the
20–40 mg L−1 and phosphorus of 1–10 mg L−1 (McGinn et al. semicontinuous culture than at the end of the batch culture
2011). Despite this limitation, it has been demonstrated (Arbib has also been observed for C. sorokiniana with the considered
et al. 2013; Cabanelas et al. 2013; Gómez-Serrano et al. 2015; flocculation agents (Escapa et al. 2015). In view of these re-
Prandini et al. 2016) that biomass productivity is compatible sults, it may be inferred that the efficiency of flocculation is
with removal of nutrients from wastewater. affected by the stage of the culture and, therefore, by the age of
The almost non-existent natural sedimentation of the microalgae. It is well known that the biochemical composition
microalgal biomass is due to a combination of the small of the cell surface varies between exponential and steady state
cell size of the microalgae (3–5 μm), their low concentra- cultures, resulting in different flocculation behaviour
tion in the culture medium (0.5–2.5 g L−1) and the elec- (Henderson et al. 2008; Danquah et al. 2009). Moreover,
trostatic repulsive forces among cells (Molina Grima et al. microalgae excrete organic matter, such as polysaccharides
2003). Therefore, it has been reported that C. vulgaris and proteins, which are accumulated in the growth medium
sedimentation velocity is lower than 10−6 m s−1, which and can compete with the algal surface for the flocculants
leads to a non-efficient recovery process (Granados et al. (Chen et al. 2010; Zhang et al. 2012b).
2012). Apart from cell size, the differences in the natural In conclusion, in this work, T. obliquus has been shown to
sedimentation between C. vulgaris and T. obliquus may have a higher growth capacity than C. vulgaris. Indeed, the
be also related with the composition of the hydrophobic removal efficiencies of both pharmaceuticals, especially of
extracellular polymeric substances (EPS) of the cells, salicylic acid, were higher using T. obliquus than using
which is an important factor for the natural sedimentation C. vulgaris in both batch and semicontinuous culture. On
of microalgae (Salim et al. 2014). the other hand, the removal of nitrates and phosphates by these
The results obtained with metal salts in previous stud- strains was similar and nearly complete at the end of the batch
ies with C. sorokiniana (Escapa et al. 2015) showed culture, T. obliquus being more efficient at the steady state of
higher efficiencies and in less time than in the present the semicontinuous culture. In spite of this, the recovery effi-
work with C. vulgaris and, especially, S. obliquus. The ciencies of C. vulgaris with the flocculants here used were
doses that proved to be more efficient for the recovery much higher than those obtained with T. obliquus, which, on
of C. sorokiniana (Escapa et al. 2015) were 200 mg g−1 the other hand, showed an ability to naturally flocculate. The
of AlCl3 (95.23 ± 0.51 %, 1 min) and 250 mg g−1 of obtained results point to the feasibility of using the microalgae
FeCl3 (98.35 ± 0.46 %, 1 min), which are close to values here considered in bioremediation systems and revealed that
101
J Appl Phycol
this type of study is the key for the selection of the strain, Granados M, Acién F, Gómez C, Fernandez-Sevilla JM, Molina Grima E
(2012) Evaluation of flocculants for the recovery of freshwater
which depends on the application. Still, further research is
microalgae. Bioresour Technol 118:102–110
needed to assess the mechanisms involved in the removal of Henderson R, Baker A, Parsons S, Jefferson B (2008) Characterisation of
pharmaceuticals by these strains, namely to determine the rel- algogenic organic matter extracted from cyanobacteria, green algae
ative importance of microalgal metabolism and adsorption. and diatoms. Water Res 42:3435–3445
Henry TB, Black MC (2008) Acute and chronic toxicity of fluoxetine
(selective serotonin reuptake inhibitor) in western mosquitofish.
Acknowledgements Authors thank University of León for funding Arch Environ Contam Toxicol 54:325–330
given to MICROTRAT (project AE429). Carla Escapa acknowledges Hom-Díaz A, Llorca M, Rodríguez-Mozaz S, Vicent T, Barceló D,
the Spanish Ministry of Education, Culture and Sports for her PhD fel- Blánquez P (2015) Microalgae cultivation on wastewater digestate:
lowship (FPU12/03073). Sergio Paniagua thanks the Spanish Ministry of β-estradiol and 17 α-ethynylestradiol degradation and transforma-
Education, Culture and Sports for his PhD fellowship (FPU14/05846). tion products identification. J Environ Manag 155:106–113
Marta Otero acknowledges University of León for the extension of the Mann J, Myers J (1968) On pigments growth and photosynthesis of
RYC-2010-05634 contract from the Spanish Ministry of Economy and Phaeodactylum tricornutum. J Phycol 4:349–355
Competitiveness, State Secretariat for Research, Development and Matamoros V, Uggetti E, Garcia J, Bayona J (2016) Assessment of the
Innovation. mechanisms involved in the removal of emerging contaminants by
microalgae from wastewater: a laboratory scale study. J Hazard
Mater 301:197–205
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103
Capítulo 7
Artículo IV
Comparative assessment of diclofenac removal from water by
different microalgae strains

Algal Research, 18 (2016): 127-134
Algal Research 18 (2016) 127–134
Algal Research
journal homepage: www.elsevier.com/locate/algal
Comparative assessment of diclofenac removal from water by different

microalgae strains
C. Escapa, R.N. Coimbra, S. Paniagua, A.I. García, M. Otero ⁎
Department of Applied Chemistry and Physics, Institute of Environment, Natural Resources and Biodiversity (IMARENABIO), University of León, 24071 León, Spain
Article history: Diclofenac has recently been included in the first watch list of substances to be monitored in all member states so
Received 11 January 2016 as to evaluate its future inclusion in the priority substances list by the Water Framework Directive. Therefore, in
Received in revised form 25 May 2016 view of upcoming limitations on diclofenac discharge, the objective of this work was to assess its removal from
Accepted 6 June 2016
water by a microalgae-based treatment. Moreover, considering microalgae application in wastewater treatment,
Available online 17 June 2016
it was aimed to verify if their nutrient removal capacity was affected by the presence of diclofenac. For a compar-
Keywords:
ison purpose, three different microalgae strains, namely Chlorella sorokiniana, Chlorella vulgaris and Scenedesmus
Emerging contaminants obliquus, were cultured in photobioreactors under identical controlled conditions. For the three strains, the addi-
NO3–N tion of diclofenac meant an organic carbon source and provided higher biomass concentration. C. sorokiniana was
PO4–P the strain showing the largest increase of growth rate and microalgae density, which were above 25% and 31%,
Wastewater treatment respectively, compared with the positive control. However, S. obliquus showed the highest efficiency in the re-
Phytoremediation moval of diclofenac (N79%) and nutrients (N87% nitrates, N 99% phosphates) per litre and per gram of biomass.
Phycoremediation These results pointed to the potential application of microalgae for the removal of pharmaceuticals in the biore-
mediation of wastewater.
© 2016 Elsevier B.V. All rights reserved.
1. Introduction were finally included in the watch list of substances to be monitored

in all member states to support future reviews of the priority substances
The occurrence of micropollutants in the aquatic environment has list.
become, during the last few decades, a global issue of increasing envi- Diclofenac (2-(2-(2, 6-dichlorophenylamino)phenyl)acetic acid) is a
ronmental concern. Micropollutants, commonly termed as emerging non-steroidal anti-inflammatory drug (NSAID). It is sold as oral tablets
contaminants (ECs), consist of a vast and expanding array of anthropo- or as a topical gel under the commercial names Acoflam, Algosenac,
genic as well as natural substances that are not currently covered by Almiral, Ana-Flex, Anthraxiton, Antiflam, Arcanafenac, Arthrex,
existing water regulations but are thought to be a threat to environmen- Arthrifen, Arthtotec, Diclabeta, Diclac, Dicloabac, Diclodoc, Diclofenac–
tal ecosystems and human health [1]. Among ECs, pharmaceuticals and Ratiopharm, Diclofenbeta, Diclomex, Diclowal, Dicuno, Difen, Diklotab,
personal care products (PPCPs) have received considerable attention Dolgit–Diclo, Eese, Effekton, Jutafenac, Monoflam, Motifene Dual,
with respect to their environmental fate and toxicological properties Rewodina, Sigafenac and Voltaren [5]. It is among the most consumed
over the last decade [2]. Pharmaceuticals represent an especially worry- drugs in the world, although its consumption varies between and with-
ing class since they were designed to cause a physiological response and in countries from 195 to 940 mg per inhabitant and year [5]. Part of the
their presence in the environment may affect non-target individuals consumed diclofenac is excreted in its original form so entering munic-
and species [3], including humans [4]. This concern has led to the recent ipal wastewater, where its concentration reflects its consumption by the
consideration by European regulations within the Water Framework Di- residents in the specific sewer system [5].
rective (2000/60/EC) (WFD). The Commission proposal of 31 January Pharmaceuticals in domestic sewage or from hospital or industrial
2012 foresaw the inclusion of three ECs, namely diclofenac, 17-beta-es- discharges end in municipal sewage treatment plants (STPs), but con-
tradiol (E2) and 17-alpha-ethinylestradiol (EE2) in the list of priority ventional wastewater treatments have been reported to be ineffective
substances. However, by the Directive 2013/39/EU, these pollutants in the removal of such pollutants, with efficiency values of b5–40% [6].
In fact, STPs were not originally designed for the removal of pharmaceu-
ticals due to the nonexistence of limiting regulations on their discharge
⁎ Corresponding author.
E-mail addresses: carla.escapa@unileon.es (C. Escapa), ricado.decoimbra@unileon.es
[7,8]. Consequently, STPs are important sources of such pollutants in the
(R.N. Coimbra), sergio.paniagua@unileon.es (S. Paniagua), ana.garcia@unileon.es aquatic environment [1,4]. In this regard, Verlicchi et al. [9], who
(A.I. García), marta.otero@unileon.es (M. Otero). reviewed the occurrence of 118 pharmaceuticals in the influent and
http://dx.doi.org/10.1016/j.algal.2016.06.008
2211-9264/© 2016 Elsevier B.V. All rights reserved.
107
128 C. Escapa et al. / Algal Research 18 (2016) 127–134
effluent of 244 STPs, found that the occurrence of some of them in the Culture Collection of Algae (Fig. 1). These microalgae strains are
effluent discharged into surface water bodies may pose a medium– among the most commonly used for wastewater treatments, have
high (acute) risk to aquatic life. Among the studied pharmaceuticals, high growth rates and are able to grow under a wide range of conditions
diclofenac was shown to have the highest average mass load (240 mg/ [17], which motivated their choice for this study.
1000 inhabitant) in the effluents of municipal STPs [9]. The removal ef- The corresponding inoculum of each strain was cultivated in 250 ml
ficiencies of diclofenac in conventional STPs have been reported to be Erlenmeyer flasks in the standard culture medium proposed by Mann
about 17% [10], which translates into relative high concentrations in and Myers [27], which is composed of (per litre of distilled water):
the respective effluents [11]. Furthermore, an increase of pharmaceuti- 1.2 g MgSO4·7H2O, 1.0 g NaNO3, 0.3 CaCl2, 0.1 g K2HPO4, 3.0 × 10−2 g
cal concentrations in receiving waters during summer due to the rela- Na2EDTA, 6.0 × 10−3 g H3BO3, 2.0 × 10−3 g FeSO4·7H2O, 1.4 × 10−3 g
tive higher contribution of STPs' discharge to the river water flow may MnCl2, 3.3 × 10−4 g ZnSO4·7H2O, 7.0 × 10−6 g Co(NO3)2·6H2O, and
be expected [12]. 2.0 × 10−6 g CuSO4·5H2O. The inoculum was kept inside a vegetal cul-
The European Union (EU) has recognized the necessity of develop- ture chamber, where growth occurred under controlled temperature
ing a strategic approach to water pollution by pharmaceutical sub-
stances, stating that until 2017 the Commission shall “propose
measures […] with a view to reducing discharges, emissions and losses
of such substances into the aquatic environment, taking into account
public health needs and the cost-effectiveness” (Directive 2013/39/
EU). Therefore, given social and political concerns at the EU about phar-
maceuticals, and, specifically about diclofenac, it is expectable that leg-
islation on its discharge will come out in the near future. However,
research on their removal is at a very incipient state with most works
on alternative to conventional treatments having been published within
the last 5 years. Recently, Rivera-Utrilla et al. [13] published a review on
treatments for the elimination of pharmaceuticals from water, and, in
the actual context, they highlighted the necessity of studying and devel-
oping sustainable solutions. With this purpose, microalgae-based
wastewater treatment technologies have emerged as a promising op-
tion [14–16].
A main advantage of microalgae application in wastewater treat-
ment is that wastewaters constitute a nutrient source for microalgae
[17–19] and one of the priority objectives in a wastewater treatment
is the removal of high concentrations of nitrates and phosphates to
meet the European Commission Directive 98/15/EEC requirements.
Thus, microalgae wastewater treatment may be an option to reduce
the costs associated to the production of microalgae based biofuels
and/or CO2 consumption [20,21]. In addition, the microalgae
phytoremediation potential is well known and the feasibility of cultivat-
ing microalgae in wastewaters to remove organic carbon and inorganic
nutrients have been extensively studied [18,19,22–24]. Among
microalgae, the most studied genuses for wastewater treatment are
Chlorella and Scenedesmus [17].
However, very few studies have been carried out on the perfor-
mance of different species for the removal of pharmaceuticals. In this
sense, Peng et al. [25] studied the biotransformation of progesterone
and norgestrel in aqueous solutions by Scenedesmus obliquus and Chlo-
rella pyrenoidosa; Escapa et al. [15] proved the capacity of Chlorella
sorokiniana to remove salicylic acid and paracetamol from aqueous so-
lution; and Hom-Díaz et al. [26] studied the removal of E2 and EE2
from anaerobic digester centrate by Selenastrum capricornutum and
Chlamydomonas reinhardtii. However, to our best knowledge the re-
moval of diclofenac from water by different microalgae species has
not been assessed yet. Thus, the present study focuses on the evaluation
and comparison of the removal of diclofenac from water by three differ-
ent microalgae species, namely Chlorella sorokiniana, Chlorella vulgaris
and Scenedesmus obliquus (homotypic synonymous of Acutodesmus
obliquus).
2. Experimental
2.1. Microorganism and culture conditions
The microalgae strains used in this study were Chlorella sorokiniana

CCAP 211/8 K (spherical cells with 3–5 μm diameter) from UTEX Culture
Collection of Algae, Chlorella vulgaris SAG 221-12 (spherical cells with 3–
5 μm diameter) from SAG Culture Collection of Algae and Scenedesmus Fig. 1. Microscopic images of microalgal cells of Chlorella sorokiniana (a), Chlorella vulgaris
obliquus SAG 276-1 (ovoid cells with 5–10 μm diameter) from SAG (b) and Scenedesmus obliquus (c).
108
C. Escapa et al. / Algal Research 18 (2016) 127–134 129
(25 ± 1 °C), irradiance in the range of photosynthetically active radia- dried in an oven at 80 °C for 24 h. Additionally, the growth of the culture
tion (175 μE m−2 s−1), photoperiod (12:12) and shaking (250 rpm). was measured as cell density (Nc) by cell counting with a Neubauer
Bubbling column photobioreactors (PBRs) with spherical bases chamber.
(40 mm diameter and 300 mm height with 300 ml capacity) were The concentration of diclofenac was analysed by a Jasco HPLC appa-
used for the experimental set-up, keeping an operating volume of ratus equipped with a PU-980 pump, a detector UV–Vis Barspec, a
250 ml. Each PBR was inoculated with the same volume of correspond- Phenomenex C18 column (5 μm, 110 Å, 250 × 4.6 mm), a Rheodyne in-
ing pre-cultured microalgae to ensure the same initial concentration in jector and a 50 μl loop. The wavelength of detection was 276.5 nm and
each triplicate, about 3 × 106 cells ml−1. the mobile phase consisted of a mixture of
The same culture conditions and culture medium used for the inoc- acetonitrile:water:orthophosphoric acid (70:30:0.1, v/v/v), which was
ulum growth were used during the experimental phase. However, dur- pumped at a flow rate of 1 ml min−1. HPLC quality acetonitrile
ing the experimental phase, the irradiance, which was supplied by 8 (CH3CN) from LAB-SCAN, orthophosphoric acid (H3PO4) from Panreac
fluorescent lamps (58 W, 2150 lm, Philips, France), was and ultrapure water obtained by a Millipore System were used for the
370 μE m−2 s−1. Moreover, during the experimental phase, the culture preparation of the mobile phase. Before use, each mixture was passed
was aerated with filtered air (0.22 μm sterile air-venting filter, through a Millipore 0.45 μm pore size filter and degasified in an ultra-
MillexFG50-Millipore), at a rate of 0.3 v/v/min, enriched with CO2 at sound bath during 30 min. Before analysis, all the samples were centri-
7% v/v, which was injected on demand to keep a constant pH (pH = fuged twice at 7500 rpm for 10 min (SIGMA 2-16P centrifuge).
7.5 ± 0.5), as controlled by a pH sensor. Nitrates (NO3–N) were measured by the ultraviolet spectrophoto-
metric screening method, according to Standard Methods 4500-NO− 3 B
2.2. Experimental set-up [28]; and phosphates (PO4–P) were measured by the ascorbic acid
method, according to the Standard Methods 4500-P E [28].
Reactors were operated in batch mode until the end of the exponen-
tial growth phase and then under semicontinuous mode until the 2.4. Data analysis
growth parameters remained constant. During the batch culture, an al-
iquot of 5 ml was daily taken from each PBR for the analytical determi- Growth kinetics were resolved in OriginPro 8 using the logistic
nations, this volume being replaced with distilled water to keep the model. This classic model was originally described by Verhulst [29]
operation volume. During the semicontinuous culture, 30% of the cul- and has been proven to fit the growth of microalgae [30]. It corresponds
ture volume was daily harvested and used for analysis, this volume to a sigmoidal curve that describes the relationship between a
being replaced with fresh medium. Experiments were carried out in microorganism's growth and density in limited environmental condi-
triplicate and simultaneously, all the PBRs under identical conditions. tions (Eq. (4)).
For each strain of microalgae two types of experiments were carried
out: a treatment with inoculated culture medium and 25 mg l− 1 K
diclofenac (with C. sorokiniana DCS, C. vulgaris DCV, S. obliquus DSO) N¼ ð4Þ
1 þ ea−rt
and a positive control with inoculated culture medium (with C.
sorokiniana CCS+, C. vulgaris CCV+, S. obliquus CSO+). Also the corre-
sponding negative control was assayed with 25 mg l−1 diclofenac in Where N (g l−1) is the algal density at time t (h), K (g l−1) is the car-
culture medium with no microalgae (CD −). Diclofenac rying capacity (the maximum algal density reached in the culture), a is a
(C14H10Cl2NNaO2) was purchased from Sigma–Aldrich (Steinheim, Ger- constant in the logistic model that indicates the relative position from
many) as diclofenac sodium salt (≥ 99%), which main properties are: the origin and indicates the duration of the lag phase, and r (d−1) is
melting point 256–257 °C, water solubility 50 mg ml− 1, flash point the specific growth rate.
203 °C, enthalpy of vapourization 70.07 kJ mol−1, boiling point 412°C Furthermore, the kinetics of the removal of diclofenac and nutrients
at 760 mm Hg, and vapour pressure 1.59 × 10−7 mm Hg at 25 °C. were fitted to the logistic model. In each case, the parameter K (g l−1) is
Throughout the experiments, the growth of the culture was daily the maximum removal capacity by the microalgae in the culture. The
monitored by biomass concentration and cell density. The removal of parameter a is a constant in the logistic model that indicates the dura-
diclofenac was daily determined by the analysis of the concentration tion of the lag phase, that is, the delay in the removal of the target com-
of this drug in the culture medium. Moreover, in order to assess the re- pound. Finally, the parameter r (d−1) is the specific removal rate.
moval of nutrients, the concentrations of nitrate and phosphate were Finally, differences among the strains with respect to the kinetic pa-
daily monitored. rameters of growth, removal of diclofenac and nutrients were compared
by a non-parametric test using IBM SPPS Statistics 21. The comparison
2.3. Analytical methods of means was performed by means of the U Mann-Whitney test. Signif-
icance was defined as p ≤ 0.05.
Biomass concentration (Cb) was determined by optical density at For the removal of diclofenac and nutrients (NO3–N and PO4–P), the
680 nm (OD680) by spectrophotometric (UV/visible spectrophotometer volumetric efficiency was calculated as the difference between the aver-
BECKMAN DU640) and verified by dry weight. Preliminary studies were age concentration of each of them in the influent (Cinf) and in the efflu-
conducted to determinate the relationship between dry weight and ent for each sampling day (Cefflu), at the daily dilution rate
OD680 for each strain; as shown in Eq. (1) for C. sorokiniana, in Eq. (2) corresponding to each operation stage (D) (Eq. (5)). During the batch
for C. vulgaris and in Eq. (3) for S. obliquus: culture these efficiencies are cumulatively expressed as mg l−1 and as
mg l−1 d−1 during the steady state of the semicontinuous culture.
ODC:S 680 ¼ 5:1834 C b þ 0:0128; R2 ¼ 0:9983 ð1Þ

2
Volumetricefficiency ¼ C inf −C efflu D ð5Þ
ODC:V 680 ¼ 2:7933 C b þ 0:0317; R ¼ 0:9958 ð2Þ
ODS:O 680 ¼ 2:0098 C b þ 0:0451; R2 ¼ 0:9915: ð3Þ The specific efficiency of the removed diclofenac and nutrients
(NO3–N and PO4–P) was calculated as the ratio between the volumetric
Dry weight measurements were performed by filtering 10 ml of cul- efficiency and the biomass concentration (Cb) (Eq. (6)). Likewise, during
ture through a 0.45 μm Whatman filter, which was then washed with the batch culture these efficiencies are expressed cumulatively as mg g
20 ml HCl (0.5 M) to dissolve precipitated salts. Then, the filtrate was biomass−1 and as mg g biomass−1 d−1 during the steady state of the
109
Fig. 2. Experimental data on growth curves of Chlorella sorokiniana (CCS+ ●, DCS ○), Chlorella vulgaris (CCV+ ▲, DCV Δ) and Scenedesmus obliquus (CSO+ ■, DSO □). Continuous lines
correspond to fittings by the logistic kinetic model during batch culture. Experiments were performed in triplicate and bars show standard derivations.
semicontinuous culture. of organic carbon. Meanwhile, differences among positive controls

may be related to the characteristics of each microalgae strain.
C inf −C efflu D Regarding microalgae growth rate, there were significant differences
Specificefficiency ¼ ð6Þ
Cb (p ≤ 0.05) between the positive control and their corresponding treat-
ment of the two strains of the genus Chlorella. However, no significant
differences (p N 0.05) were detected in the case of S. obliquus. In addi-
3. Results and discussion tion, the positive controls of each strain reached significantly different
growth rates (p ≤ 0.05). The highest r was reached by the treatment of
3.1. Growth of the culture C. sorokiniana (DCS) with diclofenac and the lowest one by the positive
control of C. vulgaris (CCV+).
The microalgae growth curves of C. sorokiniana, C. vulgaris and S. On the other hand, during the semicontinuous mode, daily dilution
obliquus and their fittings to the logistic kinetic model, including the rates produced instability and the growth rate declined throughout sev-
positive controls and the treatments with diclofenac, are represented eral days until the growth parameters remained constant during the
as values of biomass concentration versus time in Fig. 2. The differences steady state. This instability is a typical behaviour in the microalgae cul-
among the treatments were analysed according to growth kinetic pa- ture when the growth conditions change and it is related with an adap-
rameters as shown in Table 1. There were significant differences with tation phase.
respect to the parameter a (p ≤ 0.05) between the positive control and Therefore, in view of the obtained results, it may be inferred that the
the corresponding treatment of each strain of microalgae, reaching presence of diclofenac provided higher biomass concentration com-
greater values in the case of the treatments with diclofenac. Therefore, pared with the corresponding positive control of each strain and also a
the presence of diclofenac produced a delayed response in the begin- higher growth rate. However, in all cases, the addition of the target
ning of the exponential growth phase compared with the positive con- pharmaceutical produced a larger lag phase. The strain that displayed
trol. In addition, among all positive controls, only C. vulgaris showed the best biomass productivity was C. sorokiniana, which showed the
significant differences (p ≤ 0.05) with a lower value of the kinetic pa- highest growth rate. Indeed, the addition of diclofenac produced an in-
rameter a. crease above 31% in the carrying capacity and above 25% in the growth
As it can be seen in Fig. 2, the treatments with diclofenac achieved rate compared to the corresponding positive control of this strain.
higher biomass concentration than their respective positive controls, In a previous study with C. sorokiniana [15], growth rates deter-
showing in all cases significant differences (p ≤ 0.05) with respect to mined under the presence of paracetamol (r = 0.96) were equal to
the carrying capacity. The treatment with diclofenac and C. vulgaris those here obtained with the same dose of diclofenac (r = 0.96) while
(DCV) reached the highest K and the treatment with S. obliquus (DSO) a bit lower values were observed with the addition of salicylic acid
showed the lowest one. The same trend was verified for the positive (r = 0.76).
controls showing significant differences (p ≤ 0.05). The differences be- Growth rates attained in this research were mostly higher than
tween the positive control and the corresponding treatment of each those reported by other authors. Kumar et al. [31] achieved 0.1 day−1
strain may be explained by the fact that diclofenac provided a source for C. sorokiniana in Erlenmeyer flasks under 100 μE m−2 s− 1
Table 1
Experimental data (Cb0, Nc0, Cbm, Ncm) and logistic model kinetic parameters (K, a, r) determined for the growth of positive controls and treatments with diclofenac of C. sorokiniana
(CCS+, DCS), C. vulgaris (CCV+, DCV) and S. obliquus (CSO+, DSO). Results are given as average value ± standard deviation from n = 3.
CCS+ DCS CCV+ DCV CSO + DSO
Cb0 (g l−1) 0.04 ± 0.00 0.04 ± 0.00 0.23 ± 0.00 0.23 ± 0.00 0.14 ± 0.00 0.14 ± 0.00
Nc0 (cell ml−1) 3.39 × 106 3.39 × 106 3.53 × 106 3.53 × 106 3.40 × 106 3.40 × 106
Cbm (g l−1) 1.53 ± 0.11 2.28 ± 0.03 1.69 ± 0.06 2.51 ± 0.13 1.27 ± 0.04 1.40 ± 0.05
Ncm (cell ml−1) 2.49 × 108 ± 2.15 × 107 4.19 × 108 ± 4.38 × 106 7.91 × 107 ± 1.86 × 106 1.73 × 108 ± 2.15 × 107 5.15 × 107 ± 3.77 × 106 6.33 × 107 ± 3.19 × 106
a 3.31 ± 0.16 4.24 ± 0.00 2.60 ± 0.05 3.57 ± 0.12 3.30 ± 0.24 3.76 ± 0.37
K (g l−1) 1.58 ± 0.11 2.30 ± 0.03 1.96 ± 0.13 2.65 ± 0.10 1.34 ± 0.03 1.49 ± 0.05
r (d−1) 0.72 ± 0.04 0.96 ± 0.01 0.56 ± 0.00 0.74 ± 0.01 0.79 ± 0.03 0.81 ± 0.09
R2 0.9907 0.9988 0.9804 0.9915 0.9890 0.9860
110
Fig. 3. Experimental data on volumetric efficiency in the removal of diclofenac by Chlorella sorokiniana (DCS ●), Chlorella vulgaris (DCV ▲) and Scenedesmus obliquus (DSO ■) during batch
culture (a) and at the steady state of the semicontinuous culture (b). Continuous lines correspond to fittings by the logistic kinetic model during batch culture. Experiments were
performed in triplicate and bars show standard derivations.
irradiation, compared with 0.72 day−1 for CCS and 0.96 day−1 for DCS obtained for the removal parameters among the strains, at the end of
here obtained (Table 1). Domínguez Cabanelas et al. [32] described the batch culture, efficiencies above 65% for DCS, 69% for DCV and 98%
growth rates for C. vulgaris close to 0.38 day−1 with different domestic for DSO were achieved.
wastewaters in 2 l borosilicate reactors under 150 μE m−2 s−1 irradia- The average volumetric efficiencies for the diclofenac removal in the
tion, being lower than those obtained in this research, 0.56 day−1 for steady stage of the semicontinuous culture are shown in Fig. 3b. The vol-
CCV and 0.74 day−1 for DCV (Table 1). On the other hand, Arbib et al. umetric efficiency for DSO was 2.6 times higher than for DCS and 3.7
[33] reported growth rates for S. obliquus below 1 day−1 in 2 l Pyrex bot- times higher than for DCV, reaching values above 79%. Moreover, the re-
tles under 250 μE m−2 s−1 irradiation, which is a bit higher than those lation between the volumetric efficiency and the microalgae biomass is
here reported, 0.79 day−1 for CSO and 0.81 day−1 for DSO (Table 1). shown in Table 2 as specific efficiency. The results revealed that S.
obliquus removed above 3.0 times more diclofenac than C. sorokiniana
3.2. Removal of diclofenac and above 5.4 times more than C. vulgaris.
Hence, despite C. sorokiniana cells attaining a higher removal rate
The diclofenac concentration in each reactor was daily monitored due to their higher growth rate, it may be affirmed that S. obliquus
and compared with the concentration of this pharmaceutical in the neg- was the strain that reached the highest removal efficiency with more
ative control. Diclofenac concentration decreased over time in the treat- diclofenac removed either per litre or per gram of biomass. These results
ments with microalgae while no reduction was observed in the negative may be related with the specific strain characteristics, such as the cell
control. Therefore, it may be assumed that any decrease was due to the size, since C. sorokiniana had the smaller size while S. obliquus the largest
removal by microalgae. one.
The removal curves of diclofenac by each strain of microalgae and To our best knowledge there is no information available about the
the corresponding fittings to the logistic kinetic model during the removal of diclofenac by microalgae. However, in a previous study on
batch mode are displayed in Fig. 3a. For the three strains here studied, the removal of paracetamol and salicylic acid by C. sorokiniana [15], ef-
these removal curves displayed a similar trend than growth curves ficiencies above 67% for paracetamol and 73% for salicylic acid were de-
(Fig. 2), which points to the relationship between microalgae growth termined during the batch culture. On the other hand, during the steady
and diclofenac elimination. state of the semicontinuous culture efficiencies above 41% for paraceta-
Results from the fittings of the removal of diclofenac by the mol and 93% for salicylic acid were reached. These values are higher
microalgae strains here considered to the logistic model are displayed than those here obtained in the removal of diclofenac by C. sorokiniana
in Table 2. under the same hydraulic retention time.
The results related to the parameter a revealed values significantly On the other hand, Peng et al. [25] studied the biotransformation of
higher (p ≤ 0.05) for the treatment with C. sorokiniana (DCS) than for progesterone and norgestrel by S. obliquus and Chlorella pyrenoidosa
the other two treatments (DCV, DSO), which did not show significant with removals above 95% of progesterone by the two microalgae, nearly
differences between them (p N 0.05). The treatment DCS reached the complete removal of norgestrel by S. obliquus and almost 40% of
highest a, which indicated a delayed response in the removal of norgestrel by C. pyrenoidosa. Likewise, Hom-Díaz et al. [26] studied the
diclofenac compared with the other two treatments. This microalgae elimination of the hormones E2 and EE2 from anaerobic digestate
strain (C. sorokiniana) also displayed the largest lag phase, which further
supports a delay in the removal of the pharmaceutical, as compared
Table 2
with the other two strains.
Logistic model kinetic parameters (K, a, r) determined for the removal of diclofenac in the
Regarding the maximum carrying capacity, there were significant batch culture of C. sorokiniana (DCS), C. vulgaris (DCV) and S. obliquus (DSO). Volumetric
differences (p ≤ 0.05) between the treatment with S. obliquus (DSO), efficiency and specific efficiency attained in the steady state of the semicontinuous culture.
which showed the highest K value, and the other two treatments Results are given as average value ± standard deviation from n = 3.
(DCS, DCV). On the other hand, as it was above shown (Fig. 2), S. DCS DCV DSO
obliquus was the strain that reached the lowest biomass concentration
a 3.88 ± 0.62 3.23 ± 0.02 3.01 ± 0.38
at the end of the batch culture, which points to the higher efficiency of K (mg l−1) 14.55 ± 15.52 ± 22.43 ±
the S. obliquus cells, as compared with the other two strains. 0.73 0.26 0.20
Concerning the removal rate, the obtained results revealed signifi- r (d−1) 2.03 ± 0.33 1.44 ± 0.05 1.25 ± 0.19
cant differences (p ≤ 0.05) among the treatments. The quickest removal R2 0.9626 0.9755 0.9690
rate was that attained by the DCS treatment, which also reached the Volumetric efficiency (mg l−1 d−1) 2.18 ± 0.39 1.53 ± 0.32 5.66 ± 0.39
Specific efficiency (mg g biomass−1 1.73 ± 0.38 0.97 ± 0.19 5.21 ± 0.18
largest growth rate, with removal values 1.6 times higher than DAO
d−1)
and 1.4 times higher than DCV. Despite the different behaviours
111
Fig. 4. Experimental data on volumetric efficiency in the removal of nitrates by Chlorella sorokiniana (DCS ●), Chlorella vulgaris (DCV ▲) and Scenedesmus obliquus (DSO ■) treatments,
during batch culture (a) and at the steady state of the semicontinuous culture (b). Continuous lines correspond to fittings by the logistic kinetic model during batch culture.
Experiments were performed in triplicate and bars show standard derivations.
centrate by the microalgae Selenastrum capricornutum and Results from the fittings of the removal of nutrients by the strains
Chlamydomonas reinhardtii. After seven days of culture, these authors here considered to the logistic model are displayed in Table 3. The pa-
[26] determined removals above 88% for E2 and above 60% for EE2, rameter a revealed that the treatment with C. sorokiniana (DCS) was sig-
and assessed the relative importance of biodegradation and adsorption nificantly the quickest (p ≤ 0.05) to begin the removal of nitrates than
processes on their respective eliminations. Godos et al. [34] studied the the DSO treatment, not showing significant differences (p N 0.05) com-
removal of the antibiotic tetracycline in high-rate algal ponds and re- pared with the DCV treatment. However, DCS displayed the largest
ported efficiencies around 69%, which were attained both by delay in the removal of phosphates, showing significant differences
photodegradation and biosorption. Furthermore, Matamoros et al. [16] among the treatments (p ≤ 0.05). With respect to the carrying capacity,
studied the capability of microalgae-based wastewater treatment sys- no significant differences (p N 0.05) were detected in the case of nitrates.
tems to remove diclofenac, among other 25 emerging organic contami- However, in the case of phosphates, the treatment with C. vulgaris
nants. These authors [16] determined diclofenac removal efficiencies (DCV) showed the lowest K, which was significantly different
above 82% under HRT of 4 days and above 92% under HRT of 8 days dur- (p ≤ 0.05) from the two other treatments (DCS, DSO), these last not
ing the warm season (11–26 °C, on a daily average). These efficiencies showing significant differences between them (p N 0.05). Moreover,
are higher than the here obtained (Fig. 3b) under a HRT of 80 h and tem- the removal rate (r) results revealed that, for nitrates, the treatment
perature of 25 ± 1 °C. Differences must be related, at least to some ex- with C. vulgaris (DCV) displayed an r value significantly lower
tent, to the fact that microalgae monocultures were used in this work (p ≤ 0.05) than the other two treatments (DCS, DSO), which did not
while Matamoros et al. [16] worked with mixed microalgae strains show significant differences (p N 0.05) among them. For phosphates,
present in the wastewater, mostly identified as Stigeoclonium sp., dia- the treatment with C. vulgaris (DCV) also presented an r value signifi-
toms, Chlorella sp. and Monoraphidium sp. cantly lower (p ≤ 0.05) than the other treatments (DCS, DSO), the DCS
treatment displaying the largest r. In any case, at the end of the batch
culture, the removal of nutrients was nearly complete for the strains
3.3. Removal of nutrients here considered, since the end of the exponential growth phase was
caused by the depletion of nutrients.
The removal curves of nitrates and phosphates by each strain of On the other hand, during the steady state of the semicontinuous
microalgae and their fittings to the logistic model during the batch cul- culture, the removal efficiency of nitrates (Fig. 4b) was nearly 1.2
ture are shown in Figs. 4a and 5a, respectively. These curves have a sim- times lower than the removal of phosphates (Fig. 5b) for the strains
ilar trend than growth curves (Fig. 2) since microalgae growth is given here studied. The S. obliquus treatment (DSO) showed the highest re-
by the nutrient uptake. moval of nutrients, reaching efficiency values above 87% for nitrates
Fig. 5. Experimental data on volumetric efficiency in the removal of phosphates by Chlorella sorokiniana (DCS ●), Chlorella vulgaris (DCV ▲) and Scenedesmus obliquus (DSO ■) treatments,
during batch culture (a) and at the steady state of the semicontinuous culture (b). Continuous lines correspond to fittings by the logistic kinetic model during batch culture. Experiments
were performed in triplicate and bars show standard derivations.
112
Table 3
Logistic model kinetic parameters (K, a, r) determined for the removal of nitrates and phosphates in the batch culture of C. sorokiniana (DCS), C. vulgaris (DCV) and S. obliquus (DSO). Vol-
umetric efficiency and specific efficiency attained in the steady state of the semicontinuous culture. Results are given as average value ± standard deviation from n = 3.
DCS DCV DSO
a 2.19 ± 0.53 2.61 ± 0.14 3.24 ± 0.21

K (mg l−1) 734.80 ± 11.09 791.91 ± 16.98 758.26 ± 6.00
r (d−1) 0.83 ± 0.15 0.56 ± 0.09 0.84 ± 0.06
Nitrates R2 0.9585 0.9789 0.9809
Volumetric efficiency (mg l−1 d−1) 180.70 ± 15.78 146.32 ± 9.50 190.37 ± 14.15
Specific efficiency (mg g biomass−1 d−1) 116.45 ± 9.40 82.68 ± 3.61 175.39 ± 8.63
a 4.18 ± 0.16 3.59 ± 0.52 2.50 ± 0.32
K (mg l−1) 55.39 ± 0.06 48.49 ± 6.59 55.60 ± 0.59
r (d−1) 1.20 ± 0.06 0.69 ± 0.00 0.85 ± 0.02
Phosphates R2 0.9962 0.9624 0.9683
Volumetric efficiency (mg l−1 d−1) 16.18 ± 0.04 12.85 ± 0.43 16.27 ± 0.05
Specific efficiency (mg g biomass−1 d−1) 10.43 ± 0.18 7.27 ± 0.10 15.01 ± 0.51
and 99% for phosphates. On the contrary, the C. vulgaris treatment State Secretariat for Research, Development and Innovation (RYC-
(DCV) showed the lowest efficiency, reaching values close to 66% for ni- 2010-05634).
trates and 78% for phosphates. The results on the specific efficiency, the
relation between the volumetric efficiency and the microalgae biomass,
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[26] A. Hom-Díaz, M. Llorca, S. Rodríguez-Mozaz, T. Vicent, D. Barceló, P. Blánquez, ment in high-rate algal ponds, J. Hazard. Mater. 229 (2012) 446–449.
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ethynylestradiol degradation and transformation products identification, J. Environ. thetic wastewater by algae, Ecol. Eng. 28 (2006) 64–70.
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114
Capítulo 8
Artículo V
Ability of microalgae bioremediation systems to reduce the
toxicity of pharmaceuticals contaminated waters. Toxicity
assessment with zebrafish (Danio rerio) embryo bioassays
C. Escapa, T. Torres, T. Neuparth, M.M. Santos, A.I. García, M. Otero

Enviado a Water Research (marzo 2017)
Ability of microalgae bioremediation systems to reduce the toxicity of
pharmaceuticals contaminated waters. Toxicity assessment with zebrafish
(Danio rerio) embryo bioassays
C. Escapaa, T. Torresb, T. Neuparthb, M.M. Santosb,c*, A.I. Garcíaa, M. Oteroa*
a
Department of Applied Chemistry and Physics, Institute of Environment, Natural Resources and Biodiversity (IMARENABIO), University of León, 24071 León,
Spain
b
Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), University of Porto, 4050-123 Porto, Portugal
c
FCUP-Faculty of Sciences, University of Porto, Rua do Campo Alegre, 4169-007 Porto, Portugal
HIGHLIGHTS GRAPHICAL ABSTRACT
 Microalgae bioremediation
of drugs was evaluated in
terms of toxicity reduction.
 Microalgae bioremediation
was evaluated in reduced
the toxicity of contaminated
water with drugs.
 Effects of different
concentrations of drugs on
the embryo development of
zebrafish were assessed.
 Diclofenac caused more
severe effects on zebrafish
embryos than paracetamol
or salicylic acid.
 S. obliquus obtained the ABSTRACT
best results in reducing the
toxic effects of diclofenac In the recent years, the use of microalgae in the removal of pollutants from wastewater treatment plans
and salicylic acid. has gained considerable attention. However, little is known about its potential in the removal of
 Metabolic removal of these pharmaceuticals. Therefore, here we aimed at investigating the value of zebrafish embryo bioassays to
drugs did not result in toxic assess the efficiency of microalgae bioremediation systems in reducing the toxicity of several
biotransformation products.
pharmaceuticals contaminated waters. The work integrated zebrafish bioassays to assess the toxicity of
the pharmaceuticals diclofenac, paracetamol and salicylic acid and the bioremediation systems
Keywords: containing three different microalgae strains, namely Chlorella sorokiniana, Chlorella vulgaris and
Scenedesmus obliquus. Diclofenac caused more severe effects on zebrafish embryos and larvae than
Danio rerio
Bioremediation paracetamol or salicylic acid. S. obliquus was the strain that achieved the most efficient toxicity removal
Emerging contaminants in the case of diclofenac and salicylic acid. In both cases, S. obliquus was able to eliminate mortality and
Diclofenac to reduce abnormalities related to diclofenac and salicylic acid up to 30% and 100%, respectively. On
Paracetamol
Salicylic acid
the other hand, C. sorokiniana achieved the best results on paracetamol toxicity removal, reducing
abnormalities up to 36%. The obtained results showed that the metabolic removal of these
pharmaceuticals by the strains here studied did not result in toxic biotransformation products in the
effluents of the bioremediation systems and highlight the potential of microalgae bioremediation system
as a future alternative for the removal of pharmaceuticals from waste effluents.
1.Introduction The growing concern on the toxicological risks of pharmaceuticals in

the aquatic environment has led the European commission, within
Emerging contaminants (ECs) include a wide range of compounds the Water Framework Directive (2000/60/EC) (WFD), to propose in
such as human pharmaceuticals, veterinary medicines, 2012 the inclusion of three pharmaceuticals, namely diclofenac, 17-
nanomaterials and personal care products that may pose a beta-estradiol (E2) and 17-alpha-ethinylestradiol (EE2) in the watch
considerately environmental risk. Among them, pharmaceuticals list of priority substances. The EU Decision 2015/495, finally included
have received considerable attention over the last decade due to these pharmaceuticals with another estrogen (E1) and three
their environmental fate and toxicological properties (Tiedeken et al., antibiotics (azithromycin, clarithromycin and erythromycin) in the first
2017). Pharmaceuticals represent an important worrying class of watch list of substances to be monitored in all member states to
ECs since they are designed to cause physiological responses at support future revisions of the priority substances list (Barbosa et al.,
rather low concentrations and their presence in the environment may 2016).
pose lifelong threats to exposed non-target organisms (García- Pharmaceuticals present in domestic sewage, but also from
Rodríguez et al., 2014; Neuparth et al., 2014; Ribeiro et al., 2015). hospital or industrial discharges, reach the municipal wastewater
* Corresponding author: marta.otero@unileon.es

(M. Otero) & santos@ciimar.up.pt (M.M. Santos) 117
treatment plants (WWTPs), which are mostly ineffective in the 2008). Several studies have been carried out with the embryonic
removal of such pollutants, with efficiency values of <5–40% development of zebrafish to assess the toxicity of pharmaceuticals
(Rigobello et al., 2013). In fact, conventional wastewater treatments (Coimbra et al., 2015; David and Pancharatna, 2009; Macedo et al.,
in WWTPs were not originally designed for the removal of 2017; Ribeiro et al., 2015; Soares et al., 2009; Torres et al., 2016).
pharmaceuticals due to the lack of limiting regulations on their On the other hand, zebrafish embryo toxicity tests have also been
discharge (Barceló and Petrovic, 2006; Bolong et al., 2009). used to assess the efficiency of wastewater treatment on the removal
Consequently, WWTPs are important sources of such pollutants in of ECs toxicity (Galus et al., 2013; Na et al., 2016).
the aquatic environment (Gracia-Lor et al., 2017; la Farré et al., 2008; Currently, it has been proved the capability of microalgae, namely
Pal et al., 2014). Verlicchi et al (2012), who reviewed the occurrence C. sorokiniana, C. vulgaris and S. obliquus, as bioremediation
of 118 pharmaceuticals in the influent and effluent of 244 WWTPs, systems to remove pharmaceuticals from water (Escapa et al.,
found that the occurrence of some of them in the effluent discharged 2016a, 2016b, 2015). The present work aimed at giving a step
into surface water bodies may pose a medium–high (acute) risk to forward to further evaluate the efficiency of these systems on the
aquatic life. Among the studied pharmaceuticals, diclofenac showed basis of toxicological data from zebrafish embryo toxicity tests. To
the highest average mass load (240 mg/1000 inhabitant) in the the best of our knowledge, this is the first time that such a study is
effluents of municipal WWTPs (Verlicchi et al., 2012). The removal carried out, thus fostering a new approach to the assessment of
efficiency of diclofenac in conventional WWTPs have been reported microalgae-based treatments of pharmaceuticals contaminated
to be about 17% (Heberer et al., 2002), which translates into relative waters.
high concentrations in the final effluents (Ashton et al., 2004).
The use of microalgae in wastewater treatment was proposed in 2.Material and Methods
the 1960’s (Oswald et al., 1957) due to their high photosynthetic
efficiency, high growth rates, wide adaptability and high potential to 2.1.Microalgae bioremediation system
remove nutrients from wastewaters. The main advantage of using
microalgae in wastewater treatment is the potential recovery of algal The microalgae strains used in this study were Chlorella
biomass to be used as a fertilizer, as a source of products (e.g. sorokiniana (CCAP 211/8 K, UTEX Culture Collection), Chlorella
paraffin, olefin, glycerol, protein, anti-oxidant, pigment, plastic, etc.), vulgaris (SAG 221-12, SAG Culture Collection) and Scenedesmus
or as biofuel, but also the generation of an oxygenated high-quality obliquus (SAG 276-1, SAG Culture Collection). Microalgae were
effluent (Matamoros et al., 2016). In the recent years, the use of cultivated in bubbling column photobioreactors with spherical bases
microalgae in the removal of both organic and inorganic pollutants (40 mm diameter and 300 mm height with 300 ml capacity), keeping
has gained attention (Combarros et al., 2014; de Wilt et al., 2016; an operating volume of 250 ml. Each photobioreactor was inoculated
Hom-Díaz et al., 2015; Norvill et al., 2016). However, little is known with the required volume of the corresponding pre-cultured
about its potential in the removal of ECs, such as pharmaceuticals, microalgae in the standard culture medium Mann and Myers (Mann
and therefore several researchers have already claimed the and Myers, 1968) so to have the same initial concentration of cells.
necessity of further studies focus on the role of algal systems in the Then, photobioreactors were operated in batch mode until the end of
removal efficiency of ECs (Petrie et al., 2015). the exponential growth phase. For each strain, experiments were run
The generation of toxic products during biodegradation of ECs is in triplicate and under identical conditions in all the photobioreactors.
an important field of research (Lai et al., 2002; Peng et al., 2014; Further details on the experimental set-up and culture conditions may
Zhang et al., 2014). The toxicity of ECs degradation products be found elsewhere (Escapa et al., 2015).
depends both on the degradation pathway and the structure of the The removal of diclofenac, paracetamol and salicylic acid by each
parent compound (Norvill et al., 2016). However, the wide diversity of the referred strains was studied. For this purpose, each
of ECs and the difficulty on defining the degradation products in the pharmaceutical was added to the standard culture medium Mann and
WWTPs matrices impairs a better characterization of the Myers (Mann and Myers, 1968) at the initial concentration of 25,000
degradation pathways (Norvill et al., 2016). Stadler et al. (2012) µg l-1. Diclofenac (C14H10Cl2NNaO2, ≥ 99%) was supplied by Sigma–
stated that it is unrealistic to determine every possible degradation Aldrich as diclofenac sodium salt, paracetamol (C8H9NO2, ≥ 99%) by
products and their respective toxicity for all potential parent Roic Pharma and salicylic acid (C7H6O3, ≥ 99%) by Panreac.
compounds that might be present in wastewater. Nevertheless, Throughout the experiments, the removal efficiency of
apart from monitoring the removal of the parent compounds it is pharmaceuticals was determined by the analysis of the remaining
essential to evaluate the efficiency of wastewater treatments to concentration of the parent compounds in the culture medium by high
decrease the toxicity of final effluents. As concluded by Magdeburg performance liquid chromatography as described in Escapa et al.
et al. (2014), the comprehensive evaluation of the efficiency of (2016) for diclofenac and Escapa et al. (2015) for paracetamol and
wastewater treatment requires chemical analysis accompanied by salicylic acid. The obtained results on the bioremediation of these
toxicity tests to account for the range of not-detected compounds pharmaceuticals by each microalgae are summarized in Table 1 and
and, in particular, the toxicity of degradation products formed during Fig. 1.
the treatment process. At the end of the batch culture (8-10 days), the treated effluent from
Zebrafish (Danio rerio) is one of the most popular model organisms each photobioreactor was used for toxicity assessment. For the
in (eco)toxicology (Dai et al., 2014; Torres et al., 2016). It is used in toxicity bioassays, 75 ml of the corresponding homogenized effluent
several ecotoxicological test protocols and recommended as test were centrifuged twice at 5974 xg during 10 min (SIGMA 2-16P
species (Oberemm, 2000; OECD., 2013) due to its size, robustness, centrifuge) and the supernatant was preserved at 20 ± 1 ºC.
short life-cycle, large number of offspring, ease of maintenance and
reproduction in laboratory conditions. Moreover, zebrafish eggs are 2.2.Zebrafish embryo toxicity test
translucent, which allows monitoring of embryo development under a 2.2.1.Experimental solutions and effluents from the bioremediation
stereomicroscope (Soares et al., 2009). In addition the zebrafish system
embryos constitute an attractive model for environmental risk
assessment studies since they offer the possibility to perform small- In a first phase, the toxicity of experimental solutions containing the
scale, cost-effective and high-throughput analyses (Scholz et al., pharmaceuticals used in this study, namely diclofenac, salicylic acid
118
Table 1
Remaining concentration and removal efficiency of diclofenac, paracetamol and salicylic acid after microalgae bioremediation under batch operation mode.
Abbreviations given to the dilutions of these effluents and to the experimental solutions of each pharmaceutical are shown in the last column of the table.
Remaining Removal
Microalgae concentration efficiency Reference Abbreviations
(µg l-1) (%)
DCS_50 (50% dilution rate)

C. sorokiniana 6,830 ± 400 65.65 ± 2.03 (Escapa et al., 2016a)
DCS_10 (10% dilution rate)
Effluents from DCV_50 (50% dilution rate)
Diclofenac
microalgae C. vulgaris 5,960 ± 880 69.36 ± 4.51 (Escapa et al., 2016a)

DCV_10 (10% dilution rate)
bioremediation
DSO_50 (50% dilution rate)
S. obliquus 270 ± 80 98.62 ± 0.42 (Escapa et al., 2016a)
DSO_25 (25% dilution rate)
Experimental D_25000, D_12500, D_6250,
Named as D_ followed by the corresponding concentration (µ l-1)
solutions D_2500, D_250, D_25
PCS_50 (50% dilution rate)
C. sorokiniana 7,020 ± 670 67.34 ± 3.13 (Escapa et al., 2015)
PCS_25 (25% dilution rate)
Effluents from
Paracetamol
PCV_50 (50% dilution rate)

microalgae C. vulgaris 16,940 ± 40 21.88 ± 0.16 (Escapa et al., 2016b)
PCV_25 (25% dilution rate)
bioremediation
PSO_50 (50% dilution rate)
S. obliquus 12,420 ± 1,630 40.98 ± 7.72 (Escapa et al., 2016b)
PSO_25 (25% dilution rate)
Experimental P_25000, P_12500, P_6250,

Named as P_ followed by the corresponding concentration (µ l-1)
solutions P_2500, P_250, P_25
SCS_50 (50% dilution rate)
C. sorokiniana 5,580 ± 740 73.45 ± 3.53 (Escapa et al., 2015)
SCS_10 (10% dilution rate)
Effluents from
Salicylic acid
SCV_50 (50% dilution rate)

microalgae C. vulgaris 18,660 ± 900 25.34 ± 3.61 (Escapa et al., 2016b)
SCV_10 (10% dilution rate)
bioremediation
SSO_50 (50% dilution rate)
S. obliquus 1,330 ± 40 93.43 ± 0.21 (Escapa et al., 2016b)
SSO_10 (10% dilution rate)
Experimental S_25000, S_12500, S_6250,
Named as S_ followed by the corresponding concentration (µ l-1)
solutions S_2500, S_250, S_25
and paracetamol, was assessed. The concentrations of carried out, including an experimental control and a solvent control.
pharmaceuticals in these solutions were selected based on the initial All the solutions were prepared daily to ensure the concentration of
concentration of each compound in the microalgae bioremediation pharmaceutical and avoid the proliferation of microorganisms.
system assays and the removal efficiency achieved at the end of the
batch culture. The experimental solutions were obtained by dilution 2.2.2.Danio rerio fertilization and embryos collection
of each compound in freshwater (dechlorinated and aerated tap
Adults zebrafish were kept at a water temperature of 28 ± 1 ºC
water in a recirculation system with mechanical and biological filters).
under a photoperiod of 14:10 h (light:dark) in 70 litres aquaria with
A first set of experiments was carried out with 10x dilutions (25,000;
freshwater (dechlorinated and aerated tap water in a recirculation
2,500; 250; 25 µg l-1) in order to cover a wide range of concentrations.
system with both mechanical and biological filters). The adults were
A second set of experiments was carried out in order to find out if
fed ad libidum twice a day with commercial fish diet Tetramin (Tetra,
the embryo development was affected to some extent by the
Melle, Germany) supplemented with Artemia spp.
microalgae culture medium. The experimental solutions were 2x
In the afternoon before breeding, adult males and females (2:1)
dilutions of the Mann and Myers culture medium with freshwater
were housed in a cage with a bottom cover with glass marbles, within
(100%, 50%, 25%, 12.5%).
a 30 L aquarium under the same water and photoperiod conditions
A third set of experiments was carried out on to combine the toxicity
as the stock, to allow the fall of the eggs to the bottom of the
of each pharmaceutical and microalgae culture medium.
aquarium. Spawning and fertilization of the eggs were stimulated by
Experimental solutions were prepared according to the results of the
the beginning of the light period. The fertilized eggs were collected
previous assay. For each pharmaceutical, the highest concentration
from the bottom of the aquarium and were cleaned several times with
tested was 12,500 µg l-1, considering the initial pharmaceuticals
water to remove detritus and avoid the proliferation of
concentration in bioremediation system and that it was necessary a
microorganisms throughout the experiments.
50% dilution to avoid effects of the culture medium. Therefore, at this
stage, experimental solutions were done by dilution of the
2.2.3.Embryo bioassays
pharmaceutical (12,500, 6,250, 2,500, 250 µg l-1) in the culture
medium Mann and Myers at 50% with freshwater. The static-water renewal toxicological tests with zebrafish embryo
Finally, the effluents from the microalgae bioremediation system were performed according to the OECD guidelines (OECD, 1998)
were tested. These effluents, which contained the pharmaceuticals and Ribeiro et al. (2015). The cleaned eggs were observed with a
remaining concentrations indicated in Table 1, were all diluted at 50% magnifying glass and 10 fertilized eggs were placed in 24-wells filled
with freshwater, and then at 25 or 10% depending on the removal with 2 ml of freshly solutions and controls. The 24-well plates were
efficiency achieved by the corresponding system. Therefore, the incubated at 26.5 ºC during 144 h under the same conditions as the
reduction of toxicological effects achieved in the treatments by adults. The medium was daily renovated to maintain the dissolved
microalgae bioremediation will be compared with the concentration oxygen, the concentration of pharmaceuticals and to avoid the
of 12,500 µg l-1 of the corresponding parental compound, as initial proliferation of microorganisms. In addition, the dead embryos and
concentration. In all cases, six replicates of each treatment were
119
chorions after the hatching were removed at each time-point
observation. 2.2.4.Statistical analysis
The effects of the exposure were evaluated at four representative
All data were analyzed in SPPS Statistics software version 21.
periods of the embryo development: gastrula period (75%-epiboly
Normality and homogeneity of variance of the data were checked by
stage), pharyngula period (prim 15-16), larval stage (protruding-
Kolmogorov-Smirnov test and Levene test, respectively. Due to the
mouth) and juvenile at 8, 32, 80 and 144 hours post fertilization (hpf),
absence of normality, the data were subsequently analyzed by
respectively (Fig. 2).
Krustal Wallis test, followed by a multiple comparison rank test (U
Mann-Whitney test). Significance was defined at p ≤ 0.05. Statistical
analysis was carried out at 8, 32, 80 and 144 hpf for the endpoints
mortality rate, 75%-epiboly stage (8hpf), total abnormalities and
larval length (144 hpf). Control and solvent control were grouped
when no significant differences between then were detected. Also,
for each pharmaceutical compound, the treatments with the same
concentration were grouped when no significant differences between
then were detected.
To increase statistical power, all abnormalities detected were
grouped to determine the percentage of total abnormalities.
Fig. 1. Remaining concentration of pharmaceuticals during the microalgae

bioremediation system in the removal of diclofenac (a), paracetamol (b) and
salicylic acid (c) by C. sorokiniana (●), C. vulgaris () and S. obliquus ().
Experiments were performed in triplicate and bars show standard deviations.
Mortality rate and morphological abnormalities were assessed at

each time-point observation. Furthermore, 75%-epiboly stage at 8
hpf, hatching rate at 80 hpf and larval length at 144 hpf were also
evaluated in accordance with the OEDC Guidelines (OECD, 2013,
1998). The developmental stages were compared with those
described by Kimmel et al. (1995), (Fig.2.).
Morphological abnormalities were rated in abnormalities in head,
eyes, tail or yolk-sac, developmental delay, abnormality cells,
pericardial oedema, opaque chorion, excess or lack of pigmentation, Fig. 2. Periods of the embryo development of Danio rerio: gastrula period
(75%-epiboly stage) (a, b), pharyngula period (prim 15-16) (c, d) and larval
lateral position, reduced mobility and involuntary movements (Fig. 3).
stage (protruding-mouth) (e, f); from sketches (Kimmel et al., 1995) and from
The embryo development and abnormalities were observed using microscope capture.
a stereoscopic microscope inverted microscope (Nikon Eclipse .
5100T) equipped with a digital camera (Nikon D5-Fi2) and a
microscope camera controller (Nikon's Digital Sight DS-U3).
120
Fig. 3 Abnormalities detected in the experiments: abnormality cells at 8 hpf (a), developmental delay at 8 hpf (b, c), developmental delay at 32 hpf (d), lack of
pigmentation at 32 hpf (e), pericardial oedema at 32 hpf (f), several morphological abnormalities at 32 hpf (g), developmental disruption (h), several morphological
abnormalities at 80 hpf (i, j, k), morphological abnormalities in tail at 80 hpf (l), lateral position and pericardial oedema at 80 hpf (m), lateral position and
abnormalities in the yolk-sac at 80 hpf (n).
121
3. Results and Discussion on the mortality were significantly higher than control group at 80 hpf
at concentrations equal or higher than 2,500 µg l-1 and increased at
In all the set of experiments, the control group showed a mortality 144 hpf with 100% mortality at concentrations equal or higher than
rate equal or lower than 10%, which indicates that the assays were 6,250 µg l-1. Although several abnormalities were recorded at 8 hpf,
carried out in appropriate conditions (Table 2-4). at 32 hpf 100% of abnormalities were observed in the treatments with
concentrations equal or higher than 2,500 µg l-1.
3.1.Diclofenac embryo bioassays The effects of diclofenac parental compound on zebrafish embryos
have already been studied before by Ribeiro et al. (2015). The effects
The results obtained for diclofenac experimental solutions and for observed by these authors for 12,500 µg l-1 of diclofenac were less
the microalgae bioremediation system treating diclofenac severe than those found in the present study for the experimental
contaminated water are shown in Table 2. Data includes mortality solutions with the same concentration (Table 2). The mortality rate
rate, 75%-epiboly rate, total abnormalities and the main determined by Ribeiro et al. (2015) was above 18% and total
abnormalities detected at each time-point observation. Mortality rate abnormalities were above 40%, these abnormalities were mainly in
at 80 hpf is represented in Fig. 4 and the total abnormalities at 80 the yolk-sac, as in the present study.
and 144 hpf in Fig. 5.
3.1.1. Diclofenac experimental solutions 3.1.2. Effluents from the microalgae bioremediation systems treating
diclofenac contaminated water
Concerning results on the diclofenac experimental solutions, the
mortality rates at 8 hpf were not greater than 10% at any At 8 hpf, no significant differences were observed between the
concentration, however significant differences were detected in diclofenac treatments and the control concerning mortality rate, 75%-
relation to the total abnormalities. The embryos exposed to a epiboly stage or total abnormalities. However, the mortality rate at
diclofenac concentration equal or higher than 2,500 µg l-1 showed a 32 hpf was significantly increased above 17% in DCS_50 and
significant developmental delay compared with control treatment. DCV_50 treatments, in comparison with the control. Moreover, the
Therefore, the number of embryos that achieved 75%-epiboly stage solutions from C. sorokiniana (DCS_50, DCS_10) and C. vulgaris
at 8 hpf was significantly lower in the mentioned treatments, in (DCV_50, DCV_10) revealed significant embryo abnormalities at this
comparison to the control. time-point observation. All embryos from these treatments showed a
The mortality rate at 32 hpf overcame 10 % in the treatments with developmental delay and the lack of pigmentation, which incidence
concentration equal or higher than 2,500 µg l-1, showing significant ranged from 40 to 83%. Furthermore, the solutions at 50% dilution
differences in comparison to the control. Indeed, the percentage of rate showed abnormalities in the yolk-sac but in lower percentage. In
total abnormalities in these treatments was close to 100% (Table 2). the case of the S. obliquus treatments (DSO_50, DSO_10) did not
The main abnormalities detected in the treatments with diclofenac show any abnormalities at this time-point observation.
concentration of 25,000 µg l-1 and 12,500 µg l-1 were abnormalities in At 80 hpf the incidence of diclofenac on the mortality rate and the
head, eyes, tail, yolk-sac, pericardial oedema and lack of abnormalities in the yolk-sac continued to increase in DCS_50 and
pigmentation in all the embryos. The embryos exposed to DCV_50 treatments, in comparison with the control (p ≤ 0.05). The
6,250 µg l- 1 showed lack of pigmentation, and 40.7% of them also rest of the treatments did not showed any significant abnormalities at
showed abnormalities in the yolk-sac. In the concentration of this time-point observation.
2,500 µg l-1 embryos exhibited lack of pigmentation, developmental In the effluent from the bioremediation system, the increase in the
delay and 13.3% presented abnormalities in the yolk-sac. The exposure time up to 144 hpf did not yield any increase in the mortality
treatments with concentrations equal o lower than 250 µg l-1 did not rate. However, all the treatments raised the number of total
show any abnormalities. abnormalities (p ≤ 0.05), which occurrence range from 14 to 94%
The mortality rate at 80 hpf was significantly increased in the (Fig 5d). In cases, it was observed larvae with reduced mobility,
treatments with concentrations equal o higher than 2,500 µg l-1. All which showed higher incidence in the solutions with higher residual
the embryos exposed to 25,000 µg l-1 died at 80 hpf without hatching concentration of diclofenac (> 71%, DCS_50; > 65%, DCV_50).
and 81.3% not hatched embryos died in the 12,500 µg l-1 treatment. Furthermore, for these two treatments, it was observed larvae with
The mortality rate was reduced up to 36.7% in the 6,250 µg l-1 higher pigmentation than the control (> 94%, DCS_50; > 72%,
treatment and the hatching rate was increased to about 40%. In both DCV_50) and, to a lesser extent, larvae with abnormalities in the
treatments (D_12500, D_6250), all the survivors showed pericardial yolk-sac (> 44%, DCS_50; > 45%, DCV_50).
oedema and abnormalities in the yolk-sac. For 2,500 µg l-1 treatment, In summary, 12,500 µg l-1 diclofenac concentration produced
the main abnormalities detected were in the yolk-sac and affected abnormalities in head, eyes, tail, yolk-sac, pericardial oedema and
34.7% of the alive embryos. The treatments with lower lack of pigmentation on all embryos, only at 32 hpf exposure time.
concentrations showed none abnormalities (Fig. 5a). Indeed, above 81% embryos died after 48 hours and all of them at
At 144 hpf, all the embryos exposed to 12,500 µg l-1 and 144 hpf. In the case of C. sorokiniana bioremediation system, the
6,250 µg l- 1 of diclofenac died (Fig 6). The exposure to 2,500 µg l-1 removal efficiency was above 65% and the effects observed for the
produced an excess of pigmentation (> 87%) and also reduced corresponding effluents at 32 hpf were a bit lower than those
mobility of the larvae. Most of the larvae presented lateral position. expected according to their concentration (3,415 µg l-1 in DCS_50;
Despite no significant differences were detected in the mortality 683 µg l-1 in DCS_10). The incidence of the total abnormalities was
rate for the embryos exposed to 250 µg l-1 and 25 µg l-1, significant reduced above 25% in DCS_50 and above 40% in DCS_10, and only
differences were observed in the abnormalities, above 32 % of larvae caused effects on the developmental delay and the lack of
exposed to 250 µg l-1 showed reduced mobility at 144 hpf. pigmentation. In spite of the high percentage of abnormalities at 144
The exposure to the different diclofenac concentrations here tested hpf, the mortality rate was reduced above 74%. Likewise, C. vulgaris
did not affect the length of the larvae at 114 hpf (p > 0.05) (Table S.1 bioremediation system showed a similar removal efficiency of
in supplementary material). However, only the larvae exposed with diclofenac (> 69%) and also, the effects observed at 32 hpf were
concentrations equal or lower than 2,500 µg l-1 survived at this time- slightly lower than expected according to their concentration (2,980
point observation. µg l-1 in DCV_50; 596 µg l-1 in DCV_10). The percentage of embryos
The results presented here indicate that the negative effects of with abnormalities at 32 hpf was reduced above 16% in DCV_50
diclofenac are both concentration and time-dependent. The effects above 59% in DCV_10, causing the same effects as described for
122
Fig. 4 Mortality rates of Danio rerio embryos exposed to experimental solutions of diclofenac (a) and the effluents from microalgae bioremediation (b) at 80 hpf.
Control and solvent control were grouped. In addition, the treatments with the same concentration from different set of experiments (D_2500 and D_250) were
grouped. Percentages are expressed as mean ± SE (n= 12 for control, D_2500 and D_250; n= 6 exposed groups)
Fig. 5 Total abnormalities (%) of Danio rerio embryos exposed to experimental solutions of diclofenac at 80 hpf (a) and 144 hpf (b); and the embryos exposed to
the effluents from microalgae bioremediation at 80 hpf (c) and 144 hpf (d). Control and solvent control were grouped. In addition, the treatments with the same
concentration from different set of experiments (D_2500 and D_250) were grouped. Percentages are expressed as mean ± SE (n= 12 for control, D_2500 and
D_250; n= 6 exposed groups)
C. sorokiniana. The mortality rate at 144 hpf was reduced above 3.2.Paracetamol embryo bioassays
67%. The bioremediation treatment that showed the best
The overall results obtained for paracetamol parental compound
effectiveness in the reduction of the diclofenac toxicity was the
and the microalgae bioremediation paracetamol-solutions are shown
microalgae S. obliquus. The strain achieved a removal of diclofenac
in Table 3, Fig. 7 and Fig 8.
above 98% and the effluents of the corresponding bioremediation
system only revealed effects on zebrafish larvae at 144 hpf, reducing
3.2.1. Paracetamol experimental solutions
the mobility on 30.7% of them. These effects are in accordance with
the effluents concentration (135 µg l-1 in DSO_50; 67.5 µg l-1 in The mortality rates were not greater than 12% in any treatment, at
DSO_10). any time-point observation. Also, the 75%-epiboly stage and the total
The bioremediation systems by the microalgae strains here studied abnormalities at 8 hpf did not show significant differences in any
were able to reduce the concentration of diclofenac and their toxicity. treatment, in comparison to the control. At 32 hpf, lack of
The toxicity of the effluents were equivalent or lower than those pigmentation was observed on the embryos exposed to
previous determinate for the experimental solutions with the parent concentration equal or higher than 2,500 µg l-1 (p ≤ 0.05), which
compounds. According to the obtained results, it could be affirmed incidence ranged from 9.2% to 46.5%. However, no developmental
that the metabolic removal of diclofenac did not produce toxic delay was detected.
biotransformation products in none of the microalgae strains. After 48 hours, the abnormalities increased, and it was possible to
observe an excess of pigmentation on the larvae exposed to
123
paracetamol in the effluents caused the highest effects. In all the
treatments, it was observed an increased excess of pigmentation,
which ranged from 36.7% in C. sorokiniana treatment to 66.3% in
C. vulgaris treatment, at the same dilution rate. In addition, the larvae
exposed to the effluents from C. vulgaris and S. obliquus
bioremediation, showed reduced mobility, remaining in lateral
position (> 32% in PCV_50, > 22% in PSO_50).
The results have been compared with the initial concentration of
12,500 µg l-1 since the microalgae effluents were initially diluted at
50% to avoid effects of the culture medium. As it can observed in
Table 3, paracetamol concentration of 12,500 µg l-1 caused excess
of pigmentation in the 100% of the larvae at 144 hpf. Moreover,
above 61% of them remained in a lateral position and above 35%
Fig. 6 Mortality rates of Danio rerio embryos exposed to experimental also showed spams or involuntary movements. The bioremediation
solutions of diclofenac at 144 hpf. Control and solvent control were grouped. treatment that showed the best effectiveness in reducing the
In addition, the treatments with the same concentration from different set of paracetamol toxicity was C. sorokiniana, with removal efficiency
experiments (D_2500 and D_250) were grouped. Percentages are expressed
as mean ± SE (n= 12 for control, D_2500 and D_250; n= 6 exposed groups) above 67%. This efficiency was reflected in the reduction above 63%
of the total abnormalities at 144 hpf in comparison with P_12500, and
concentrations equal or higher than 2,500 µg l-1 (p ≤ 0.05), which only caused effects on the pigmentation. These results are in
incidence ranged from 10.2% to 63.0%. However, paracetamol did accordance with the expected effects due the paracetamol
not affect the hatching rate of any treatment. At 144 hpf, the effects concentration in the effluents (3,510 µg l-1 in PCS_50; 1,755 µg l-1 in
of the pharmaceutical on total abnormalities increased significantly in PCS_25). The less effective bioremediation treatment was obtained
comparison with control (p ≤ 0.05) (100% in the P_25000 treatment). by C. vulgaris, which only reduced the abnormalities occurrence
Moreover, a reduced percentage of larvae remained in lateral about 33% at 144 hpf due to the reduced removal efficiency of
position. Indeed, the larvae exposed to the highest concentrations of paracetamol (approximately 21%). The percentage of larvae that
paracetamol and which remained in lateral position, also showed showed excess of pigmentation and lateral position was in
spams or involuntary movements (> 35% in P_25000, > 25% in accordance with paracetamol concentrations (8,470 µg l-1 in PCV_50;
P_12500). 4,235 µg l-1 in PCV_25). On the other hand, the results obtained by
Exposure to the highest paracetamol concentrations caused a S. obliquus revealed an intermediate effectiveness among the three
marginal increase in the larvae length (p > 0.05) (> 4.6% in P_25000, microalgae strains. The removal efficiency achieved for paracetamol
> 4.7% in P_12500, > 3.0% in P_6250). (Table S.1 in supplementary was above 40%. Similarly, the incidence on the abnormalities was
material). reduced above 42% at 144 hpf in comparison with the initial
In view of the results of paracetamol, it is evident that the effects concentration. Exposed larvae showed excess of pigmentation and
on zebrafish embryo increased with the concentration of the lateral position, which was in accordance with the residual
pharmaceutical and throughout the time. The exposure to different paracetamol concentration (6,210 µg l-1 in PSO_50; 3,105 µg l-1 in
concentrations of paracetamol did not cause effects on the mortality PSO_25).
at any time-point observation. In spite of the abnormalities did not The bioremediation systems by the microalgae strains here studied
appear until 32 hpf, they significantly increased at 144 hpf. However, were able to reduce the concentration of paracetamol and their
at 32 and 80 hpf paracetamol only caused effects on the toxicity. Similar to diclofenac, the toxicity of the effluents could be
pigmentation. predicted based on the toxicity of the experimental solutions. This
The effects of paracetamol on the embryonic development of result highlights the effectiveness of the microalgae strains tested
zebrafish was also studied by David and Pancharatna (2009). here and the lack of production of toxic metabolites.
However, the concentrations studied by these authors (ranging from
1 to 100 µg l-1) were much lower than those here tested. In any case,
3.3.Salicylic acid embryo bioassays
the effects observed by David and Pancharatna (2009) on the
mortality rate were high (> 50% at 100 µg l-1). These authors found The results obtained for salicylic acid parental compound and the
that distribution of pigment was dose-dependent, but detected a lack microalgae bioremediation salicylic acid-solutions are shown in
of pigmentation at high relative doses of paracetamol. In addition, Table 4, Fig. 9 and Fig. 10.
larvae exposed to the highest paracetamol doses by these authors
showed altered swimming behaviour such as vibratory/shivering, in 3.3.1. Salicylic acid experimental solutions
the same way that in the present study.
Salicylic acid caused a significant increase of the mortality rate at
3.2.2. Effluents from the microalgae bioremediation systems treating 8 hpf on the embryos exposed to concentrations equal or higher than
paracetamol contaminated water 12,500 µg l-1, in comparison with the control. The other treatments
showed a mortality rate lower than 10%. However, the 75%-epiboly
Regarding the microalgae bioremediation effluents no effects on rate and the total abnormalities at 8 hpf showed significant
the mortality rate were observed at any time (Table 3), as occurred differences in the treatments with 6,250 µg l-1 concentration. The
with the paracetamol experimental solutions. Neither the 75%- main abnormalities detected were developmental delay and
epiboly rate at 8 hpf nor the hatching rate at 80 hpf were altered in abnormal cells on the embryos, up to 31.5% in S_25000 treatment.
comparison to the control. At 32 hpf it was observed lack of In the other treatments, the salicylic acid only caused abnormalities
pigmentation on the embryos, which was not greater than 15% in any in less than 5% of the embryos.
treatment (p ≤ 0.05). After 24 hours, the percentage of embryos that The mortality rate at 32 hpf was significantly higher in the
presented abnormalities, namely excess of pigmentation, increased. treatments with concentrations equal or higher than 6,250 µg l-1,
The C. vulgaris and S. obliquus treatments at 50% dilution rate reaching levels above 30% in the embryos of the S_25000 treatment.
showed the highest effects (> 38% in PCV_50, > 29% in PSO). Significant abnormalities were detected from 2,500 µg l-1
However, it was at 144 hpf when the residual concentration of concentration; in all these treatments, the embryos showed a
124
Fig. 7 Mortality rates of Danio rerio embryos exposed to experimental solutions of paracetamol (a) and the effluents from microalgae bioremediation (b) at 80 hpf.
Control and solvent control were grouped. In addition, the treatments with the same concentration from different set of experiments (P_2500 and P_250) were
grouped. Percentages are expressed as mean ± SE (n= 12 for control, D_2500 and D_250; n= 6 exposed groups)
Fig. 8 Total abnormalities (%) of Danio rerio embryos exposed to experimental solutions of paracetamol compound at 80 hpf (a) and 144 hpf (b); and the embryos
exposed to the effluents from microalgae bioremediation at 80 hpf (c) and 144 hpf (d). Control and solvent control were grouped. In addition, the treatments with
the same concentration from different set of experiments (P_2500 and P_250) were grouped. Percentages are expressed as mean ± SE (n= 12 for control, P_2500
and P_250; n= 6 exposed groups)
developmental delay and lack of pigmentation from 40.2% in S_2500 rate at any of the tested concentrations, 100% of embryos hatched
to 100% on the embryos of S_25000 treatment. Moreover, the (results not shown) in all cases.
embryo chorions exposed to 25,000 µg l-1 and 12,500 µg l-1 salicylic At 144 hpf, the mortality rate and the total abnormalities did not
acid concentration became opaque at 32 hpf (> 55% and > 38%, increase in comparison to 80 hpf. However, besides the excess of
respectively). pigmentation, some larvae remained in lateral position, but this effect
After 80 hours of exposure to salicylic acid, significant effects were was only significant (> 25%) for S_25000. The exposure to the
detected for 2,500 µg l-1 in mortality rate and for 250 µg l-1 in total different salicylic acid concentrations here tested did not affect the
abnormalities (mainly an excess of pigmentation). Therefore, when larval length at 144 hpf (p > 0.05) (Table S.1 in supplementary
increasing the exposure time, effects on the embryos were detected material).
for lower concentrations than at 32 hpf. The mortality rate increased According to the salicylic acid experimental solutions results, it is
to 55% or higher on larvae exposed to the highest treatment clearly noted that the effects associated to this pharmaceutical
concentration. The total abnormalities in S_25000 and S_12500 increased with concentration and throughout exposure time.
treatments were close to 95%. However, only the 60.4% of the However, the effects on the mortality and abnormalities were not
embryos exposed to 6,250 µg l-1 of salicylic acid showed excess of increased from 80 hours of exposure. In spite of the abnormalities
pigmentation. Moreover, the salicylic acid did not affect the hatching observed at 8 hpf, the abnormalities registered at 32 hpf were higher
125
than 90% in the treatments with concentrations equal or higher than dilution rate (> 19%), which were mainly related to a developmental
12,500 µg l-1. Indeed, these chorions became opaque and more delay. At 32 hpf, the differences in the mortality rate were maintained
sensitive to manipulation. in the SCV_50 (> 28%) and SCV_10 treatments (> 20%), in
comparison to the control. However, the greatest incidence in the
3.3.2. Effluents from the microalgae bioremediation systems treating abnormalities percentage at 32 hpf was observed in the embryos
salicylic acid contaminated water exposed to C. vulgaris effluent at 50 % (> 90%) and 10% dilution rate
(> 33%), and C. sorokiniana effluent at 50 % dilution rate (> 37%).
Concerning the results for the effluents from the microalgae
The abnormalities detected were developmental delay and lack of
bioremediation of salicylic acid (Table 4), at 8 hpf there were no
pigmentation. In the rest of the cases, only a developmental delay
significant differences between the C. sorokiniana and S. obliquus
(< 13%) was detected.
treatments in regard to the mortality rate, 75%-epiboly rate or total
At 80 hpf, the mortality rate showed the highest incidence on the
abnormalities, in comparison to the control. However, the C. vulgaris
larvae exposed to C. vulgaris effluent at 50% (> 45%) and 10%
treatments showed a significantly higher mortality rate (> 15%,
dilution rate (> 27%) and C. sorokiniana effluent at 50% dilution rate
SCV_50; > 10% SCV_10) and lower 75 %-epiboly rate (< 69%,
(> 18%) (p ≤ 0.05). The larvae exposed to these effluents also
SCV_50; < 89% SCV_10) than the control. In addition, the
showed an excess of pigmentation, which reached above 63% in the
abnormalities were significantly higher in the treatment at 50%
SCV_50 treatment.
Fig. 9 Mortality rate of Danio rerio embryos exposed to experimental solutions of salicylic acid (a) and the effluents from microalgae bioremediation (b) at 80 hpf.
Control and solvent control were grouped. In addition, the treatments with the same concentration from different set of experiments (S_2500 and S_250) were
grouped. Percentages are expressed as mean ± SE (n= 12 for control, S_2500 and S_250; n= 6 exposed groups)
Fig. 10 Total abnormalities (%) of Danio rerio embryos exposed to experimental solutions of salicylic acid at 80 hpf (a) and 144 hpf (b); and the embryos exposed
to the effluents from microalgae bioremediation at 80 hpf (c) and 144 hpf (d). Control and solvent control were grouped. In addition, the treatments with the same
concentration from different set of experiments (S_2500 and S_250) were grouped. Percentages are expressed as mean ± SE (n= 12 for control, S_2500 and
S_250; n= 6 exposed groups)
126
Table 2
Effects of diclofenac experimental solutions and microalgae bioremediation effluents on Danio rerio embryos at 8 hpf, 32 hpf, 80 hpf and 144 hpf.
Control and solvent control were grouped. In addition, the treatments with the same concentration from different set of experiments (D_2500 and D_250) were
grouped. Significant differences from control (p ≤0.05) are marked with a symbol (*) and bold. Data are expresses as mean ± SE (n= 12 for control, D_2500 and
D_250; n= 6 exposed groups)
127
Table 3
Effects of paracetamol experimental solutions and microalgae bioremediation effluents on Danio rerio embryos at 8 hpf, 32 hpf, 80 hpf and 144 hpf.
Control and solvent control were grouped. In addition, the treatments with the same concentration from different set of experiments (P_2500 and P_250) were
grouped. Significant differences from control (p ≤0.05) are marked with a symbol (*) and bold. Data are expresses as mean ± SE (n= 12 for control, P_2500 and
P_250; n= 6 exposed groups
128
Table 4
Effects of salicylic acid experimental solutions and microalgae bioremediation effluents on Danio rerio embryos at 8 hpf, 32 hpf, 80 hpf and 144 hpf
.
Control and solvent control were grouped. In addition, the treatments with the same concentration from different set of experiments (S_2500 and S_250) were
grouped. Significant differences from control (p ≤0.05) are marked with a symbol (*) and bold. Data are expresses as mean ± SE (n= 12 for control, S_2500 and
S_250; n= 6 exposed groups).
129
As occurred with the salicylic acid experimental solutions, at these compound by the strains here studied does not seem to led to
144 hpf neither the mortality rate nor the total abnormalities the production of toxic biotransformation products into the water, at
increased from values obtained at 80 hpf. However, the SCV_50 least to this experimental animal model.
treatment also showed embryos that remained in a lateral position To our best knowledge, there is not information available about the
(> 6.7%). In the other treatments no abnormalities were observed toxicological effects of transformation products of diclofenac,
and the mortality rate was lower than 12%, either at 80 hpf or paracetamol and salicylic acid after bioremediation process by
at144 hpf. microalgae on the embryonic development of Danio rerio.
As it can observed in Table 4, experimental solutions with salicylic
acid concentration of 12,500 µg l-1 caused developmental delay and 4. Conclusions
lack of pigmentation in more than 91% of the embryos and opaque
chorion in more than 38% of the embryos, just at 32 hpf exposure The bioremediation systems by the microalgae strains here studied
time. Indeed, above 41% of the embryos died at 80 hpf and above were able to reduce the concentration of diclofenac, paracetamol and
94% developed excess of pigmentation. The bioremediation system salicylic acid and their toxicity. Diclofenac caused more severe
that showed the best effectiveness in reducing the salicylic acid effects on zebrafish embryos and larvae than paracetamol or salicylic
toxicity was S. obliquus, which had shown a removal efficiency of acid. However, S. obliquus reduced the diclofenac concentration in
salicylic acid above 93% (Table 1). This efficiency was clearly the effluent and was able to eliminate the mortality and reduce the
reflected in the results obtained. The mortality rate did not show abnormalities up to 30% at 144 hpf. In the case of the salicylic acid,
significant differences in comparison with the control at any time- S. obliquus was also the strain that obtained the best results in
point observation. Also, the abnormalities detected did not reveal reducing the toxicological effects. The strain effectively eliminate the
significant differences in comparison to the control, which are in mortality and all the abnormalities. Moreover, C. sorokiniana was
accordance to their concentration (665 µg l-1 in SSO_50; 133 µg l-1 in also able to reduce the toxicological effects of salicylic acid, but a
SSO_10). Therefore, the abnormalities were reduced above 97% by mortality rate above 18% was recorded. On the other hand, none of
S. obliquus at 80 hpf. On the other hand, the results obtained with C. the microalgae strain was able to eliminate the effects of paracetamol
sorokiniana revealed an intermediate efficiency among the three on zebrafish embryos or larvae. Still, C. sorokiniana achieved the
microalgae strains here used. The removal efficiency of salicylic acid best results reducing the abnormalities up to 36%. The metabolic
achieved by C. sorokiniana was above 73% (Table 1). The mortality removal of these drugs by the strains used in this study did not
rate of SCS_50 only showed significant differences at 80 and resulted in toxic biotransformation products. These findings highlight
144 hpf, in comparison to the control, which is in agreement with the the potential of microalgae bioremediation system as a future
effluent concentration (2,790 µg l-1). Moreover, only 6.4% of the alternative for the removal of pharmaceuticals from waste effluents
larvae showed excess of pigmentation at 144 hpf and therefore, the by biological methods.
effects of salicylic acid on the abnormalities were reduced above
93%. Finally, the microalgae bioremediation system that showed Acknowledgments
lower efficiency was C. vulgaris, with removal rate of salicylic acid of
approximately 25% (Table 1). This effluent showed above 90% of Carla Escapa and Sergio Paniagua acknowledge the Spanish
embryos with lack of pigmentation in SCV_50 and above 33% in Ministry of Educations, Culture and Sports for their PhD fellowships
SCV_10 at 32 hpf, values that are in accordance to the effluents (FPU12/03073 and FPU14/05846, respectively). Marta Otero
salicylic acid concentration (9,330 µg l-1 in SCV_50; 1,866 µg l-1 in acknowledges University of León for the extension of her RYC‐2010‐
SCV_10). Moreover, the total abnormalities were reduced above 05634 contract. Authors thank University of León for funding given to
32% at 80 hpf. MICROTRAT (project UXXI2016/00128). T. Neuparth was supported
The bioremediation systems by the microalgae strains here studied by Postdoctoral fellowships from the Portuguese Science and
were able to reduce the concentration of salicylic acid and their Technology Foundation (FCT), refs. SFRH/BPD/77912/2011.
toxicity. The toxicity of the effluents were equivalent or lower than
those previous determinate for the experimental solutions with the
same concentration. In view of these results, it could be affirmed that
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131
SUPPLEMENTARY MATERIAL
Table S1. Danio rerio larval length (µm) at 144 hpf for experimental solutions and microalgae
bioremediation effluents.
Diclofenac Paracetamol Salicylic acid
Control 3850.50 ± 66.85 Control 3861.23 ± 65.84 Control 3850.50 ± 66.85

D_25000 † P_25000 4047.67 ± 78.22* S_25000 3857.33 ± 35.60
D_12500 † P_12500 4052.83 ± 44.20* S_12500 3874.83 ± 33.16
Experimental
D_6250 † P_6250 3980.83 ± 89.77* S_6250 3880.17 ± 33.03
solutions
D_2500 99.75 ± 20.28 P_2500 3909.25 ± 68.96 S_2500 3893.17 ± 46.81
D_250 106.25 ± 20.80 P_250 3900.42 ± 49.33 S_250 3903.67 ± 38.20
D_25 3852.33 ± 63.45 P_25 3874.83 ± 69.40 S_25 3939.17 ± 20.61
Control 3846.44 ± 57.65 Control 3856.88 ± 45.84 Control 3824.28 ± 65.46
DCS_50 3828.97 ± 97.53 PCS_50 3862.85 ± 54.11 SCS_50 3928.75 ± 108.70
Effluents from DCS_10 3831.79 ± 94.64 PCS_25 3869.60 ± 28.80 SCS_10 3970.68 ± 37.50
microalgae DCV_50 3900.83 ± 126.83 PCV_50 4067.03 ± 35.16* SCV_50 3900.18 ± 12.54
bioremediation DCV_10 3915.08 ± 4.94 PCV_25 3890.21 ± 22.28 SCV_10 3851.92 ± 49.97
DSO_50 3929.88 ± 76.11 PSO_50 4027.35 ± 42.54* SSO_50 3812.26 ± 88.36
DSO_25 3912.14 ± 9.10 PSO_25 3911.49 ± 17.80 SSO_10 3817.59 ± 86.16
Control and solvent control were grouped. In addition, the treatments with the same concentration
from different set of experiments (_2500 and _250) were grouped. Significant differences from
control (p ≤0.05) are marked with a symbol (*) and bold. Data are expresses as mean ± SE (n= 12 for
control, _2500 and _250; n= 6 exposed groups).
133
Capítulo 9
Resumen de resultados y discusión

9.1. CRECIMIENTO DE LAS MICROALGAS DURANTE LA BIORREMEDIACIÓN DE AGUAS

CONTAMINADAS CON FÁRMACOS
La influencia de la presencia de paracetamol, ácido salicílico y diclofenaco en el crecimiento

de las microalgas C. sorokiniana, C. vulgaris y S. obliquus durante la biorremediación de aguas
contaminadas se llevó a cabo comparando el crecimiento de los tratamientos con fármacos con los
controles positivos de cada especie. La concentración de la biomasa se determinó por densidad
óptica y se verificó mediante peso seco, mientras que la concentración celular fue determinada
mediante contaje celular. Los ensayos se llevaron a cabo en modo discontinuo y semicontinuo en
fotobiorreactores bajo condiciones controladas.
Los resultados sobre la concentración de la biomasa de cada especie de microalga a lo largo

del cultivo para cada tratamiento y control positivo, se representan en las Figuras 9.1, 9.2 y 9.3
para el paracetamol, ácido salicílico y diclofenaco, respectivamente. Así mismo, la Tabla 9.1 recoge
los parámetros cinéticos de ajuste sobre el crecimiento de cada especie. Las curvas de crecimiento
de C. sorokiniana, C. vulgaris y S. obliquus durante el cultivo discontinuo, mostraron un típico
crecimiento sigmoideo tanto en el caso de los tratamientos con fármacos como en los controles,
teniendo una duración de 8‐10 días antes de alcanzar el estado estacionario. Por otra parte,
durante el cultivo semicontinuo, las diluciones diarias generaron inestabilidad y la tasa de
crecimiento disminuyó durante varios días, antes de que los parámetros de crecimiento
permanecieran constantes durante el estado estacionario. Esta inestabilidad es una respuesta
típica en el crecimiento de microalgas cuando las condiciones de cultivo cambian, estando
relacionado con una fase de adaptación del cultivo (Figuras 9.1‐9.3).
9.1.1. Efectos del paracetamol sobre el crecimiento
La presencia de paracetamol supuso un retraso significativo del comienzo de la fase

exponencial de crecimiento de las especies del género Chlorella estudiadas, en comparación con el
control positivo, como se puede observar en los valores del parámetro a de la Tabla 9.1.
Al final del cultivo discontinuo, se detectó un incremento de la concentración de biomasa en

presencia de paracetamol en comparación con el control positivo en el caso de C. sorokiniana
(> 49%) y C. vulgaris (> 31%), como se muestra en la Figura 9.1, lo que se confirma por los valores
del parámetro K de la Tabla 9.1. Sin embargo, el cultivo de S. obliquus no vio modificada la
concentración de biomasa por la presencia de paracetamol, siendo el valor alcanzado similar al
del control positivo. Los resultados obtenidos respecto a la tasa de crecimiento (r) tan solo
137
CAPÍTULO 9
mostraron diferencias significativas en el caso C. vulgaris, resultando incrementada por la

presencia de paracetamol respecto al control positivo (Tabla 9.1).
Figura 9.1 Curvas de crecimiento de C. sorokiniana (CCS+ ●, PCS ), C. vulgaris (CCV+ , PCV □) y S. obliquus
(CSO+ , PSO ) para los tratamientos con paracetamol y controles positivos. Los puntos representan los datos
experimentales y las líneas continuas los ajustes a la cinética del modelo logístico durante el cultivo discontinuo. Los
experimentos se llevaron a cabo por triplicado, mostrando las barras de error la desviación estándar. Nota: los datos
experimentales obtenidos en el cultivo semicontinuo aparecen unidos por líneas discontinuas.
9.1.2. Efectos del ácido salicílico sobre el crecimiento
La presencia de ácido salicílico supuso un desfase en el comienzo del crecimiento

exponencial en los cultivos de C. sorokiniana y C. vulgaris, en comparación con el control positivo,
tal y como se puede apreciar en los valores del parámetro a de la Tabla 9.1.
Como se puede observar en la Figura 9.2, la concentración máxima de biomasa alcanzada al

final del cultivo discontinuo fue mayor en los tratamientos con ácido salicílico que los respectivos
controles positivos, como puede comprobarse además por el parámetro K de la Tabla 9.1. Al final
del cultivo discontinuo, el tratamiento con C. sorokiniana incrementó la concentración de biomasa
en más de un 52%, C. vulgaris en más de 18% y S. obliquus en más de un 36%, sobre sus
respectivos controles positivos. Sin embargo, la capacidad de carga en el tratamiento con ácido
salicílico en el cultivo de S. obliquus fue significativamente mayor que la alcanzada para los
tratamientos en los cultivos de C. sorokiniana y C. vulgaris. Por otra parte, la tasa de crecimiento
de C. vulgaris fue duplicada por la presencia de ácido salicílico en comparación con su control
positivo. Sin embargo, fue significativamente reducida en el caso de C. sorokiniana (reducción
> 18%) y S. obliquus (reducción > 37%).
138
Figura 9.2 Curvas de crecimiento de C. sorokiniana (CCS+ ●, SCS ), C. vulgaris (CCV+ , SCV □) y S. obliquus
(CSO+ , SSO ) para los tratamientos con ácido salicílico y controles positivos. Los puntos representan los datos
9.1.3. Efectos del diclofenaco sobre el crecimiento
La presencia de diclofenaco supuso un retraso significativo en el comienzo de la fase de

crecimiento exponencial de las tres especies en comparación con sus respectivos controles
positivos, lo que confirman los valores del parámetro a en la Tabla 9.1.
Como puede observarse en la Figura 9.3, los tratamientos con diclofenaco alcanzaron una
concentración de biomasa significativamente mayor que sus respectivos controles positivos.
Al final del cultivo en modo discontinuo, el tratamiento con C. sorokiniana mostró un incremento
de la concentración de la biomasa superior al 45%, C. vulgaris superior al 35% y S. obliquus
superior al 11%, sobre sus respectivos controles positivos (Tabla 9.1). Por otra parte, las tasas de
crecimiento de las especies del género Chlorella estudiadas fueron significativamente mayores en
comparación con el control positivo, siendo el incremento en ambos casos superior al 32%.
Sin embargo, no se detectaron diferencias significativas en las tasas de crecimiento de S. obliquus
(Tabla 9.1).
139
CAPÍTULO 9

Figura 9.3 Curvas de crecimiento de C. sorokiniana (CCS+ ●, DCS ), C. vulgaris (CCV+ , DCV □) y S. obliquus
(CSO+ , DSO ) para los tratamientos con diclofenaco y controles positivos. Los puntos representan los datos
A la vista de los resultados obtenidos, se puede deducir que la presencia tanto de

paracetamol, como de ácido salicílico o diclofenaco afecta a los parámetros de crecimiento de las
especies aquí estudiadas. En la mayoría de los tratamientos, la presencia del fármaco supuso un
incremento de la concentración de biomasa, lo cual se puede explicar por el hecho de que estos
fármacos constituyeron una fuente adicional de carbono, en forma orgánica. Es sabido que las
especies del género Chlorella y Scenedesmus pueden tener un metabolismo mixotrófico.
Sin embargo, S. obliquus no mostró un incremento significativo de la biomasa por la presencia de
paracetamol o diclofenaco, lo que sugiere que el carbono no fue utilizado como fuente de energía
para el crecimiento de las microalgas.
Las tasas de crecimiento obtenidas en esta tesis fueron, en su mayoría, superiores a las
obtenidas por otros autores. Kumar et al. (2014) lograron tasas de crecimiento de 0,1 d‐1 para
C. sorokiniana cultivada en el medio de cultivo TAP suplementado con ácido acético como fuente
de carbono. Wang et al. (2015) determinaron tasas de crecimiento menores de 0,4 d‐1 para
C. vulgaris y S. obliquus, cultivadas bajo diferentes fotoperiodos en aguas residuales domésticas
estériles. Zhao et al. (2015) determinaron tasas de crecimiento de 0,36 d‐1 para C. vulgaris y
0,45 d‐1 para S. obliquus, cultivadas en efluentes domésticos esterilizados. Cabanelas et al. (2013)
obtuvieron tasas de crecimiento para C. vulgaris cerca de 0,38 d‐1 en diferentes aguas residuales
domésticas. Por otra parte, los resultados obtenidos por Arbib et al. (2013) para S. obliquus
cultivada en aguas residuales urbanas, alcanzaron tasas de crecimiento de 1 d‐1. Contrariamente,
las tasas de crecimiento alcanzadas por S. obliquus y C. pyrenoidosa en el medio de cultivo artificial
HB‐4 (Zhang et al., 2012) fueron superiores (1.68 d‐1 en ambos casos) a las determinadas en esta
140


tesis. Sin embargo, estas tasas fueron reducidas a 1,2 d‐1 tras la adición de 10 mg l‐1 de
carbamazepina (Zhang et al., 2012). Por el contrario, en el presente trabajo, la presencia de
25 mg l‐1 de paracetamol o diclofenaco en el agua supuso un incremento de las tasas de
crecimiento de las tres especies consideradas en comparación con los respectivos controles
positivos. Mientras que en el caso del ácido salicílico (25 mg l‐1) solo supuso un incremento de la
tasa de crecimiento en el caso de C. vulgaris.
Tabla 9.1 Parámetros cinéticos del ajuste al modelo logístico (a, K, r) durante el cultivo en modo discontinuo,
determinados para el crecimiento de los controles positivos y tratamientos con paracetamol y C. sorokiniana
(CCS+, PCS), C. vulgaris (CCV+, PCV) y S. obliquus (CSO+, PSO); controles positivos y tratamiento con ácido salicílico y
C. sorokiniana (CCS+, SCS), C. vulgaris (CCV+, SCV) y S. obliquus (CSO+, SSO); y controles positivos y tratamientos con
diclofenaco y C. sorokiniana (CCS+, DCS), C. vulgaris (CCV+, DCV) y S. obliquus (CSO+, DSO). Nota: n=3.
CCS+ PCS CCV+ PCV CSO+ PSO

PARACETAMOL
a 3,77 ± 0,01 4,33 ± 0,21 4,47 ± 0,25 5,58 ± 0,03 5,45 ± 0,43 4,97 ± 0,31
K (g l‐1) 1,40 ± 0,29 2,09 ± 0,02 2,60 ± 0,15 3,42 ± 0,15 3,46 ± 0,08 3,27 ± 0,15
r (d‐1) 0,94 ± 0,06 0,96 ± 0,07 0,84 ± 0,06 1,08 ± 0,04 1,16 ± 0,07 1,12 ± 0,03
R2 0,9935 0,9939 0,9971 0,9968 0,9874 0,9886
CCS+ SCS CCV+ SCV CSO+ SSO

ÁCIDO SALICÍLICO
a 3,77 ± 0,01 4,16 ± 0,48 4,47 ± 0,25 7,99 ± 0,41 5,45 ± 0,43 4,20 ± 0,09
K (g l‐1) 1,40 ± 0,29 2,14 ± 0,13 2,60 ± 0,15 3,09 ± 0,23 3,46 ± 0,08 4,71 ± 0,30
r (d‐1) 0,94 ± 0,06 0,77 ± 0,12 0,84 ± 0,06 1,69 ± 0,13 1,16 ± 0,07 0,72 ± 0,01
R2 0,9935 0,9912 0,9971 0,9929 0,9874 0,9883
CCS+ DCS CCV+ DCV CSO+ DSO

DICLOFENACO
a 3,31 ± 0,16 4,24 ± 0,00 2,60 ± 0,05 3,57 ± 0,12 3,30 ± 0,24 3,76 ± 0,37
K (g l‐1) 1,58 ± 0,11 2,30 ± 0,03 1,96 ± 0,13 2,65 ± 0,10 1,34 ± 0,03 1,49 ± 0,05
r (d‐1) 0,72 ± 0,04 0,96 ± 0,01 0,56 ± 0,00 0,74 ± 0,01 0,79 ± 0,03 0,81 ± 0,09
R2 0,9907 0,9988 0,9804 0,9915 0,9890 0,9860
Abreviaturas: a= constante del modelo logístico, K= capacidad de carga, r= tasa de crecimiento, R2= coeficiente de
correlación.
141

CAPÍTULO 9

9.2. ELIMINACIÓN DE NUTRIENTES DURANTE LA BIORREMEDIACIÓN DE AGUAS
CONTAMINADAS CON FÁRMACOS
El estudio de la influencia de la presencia de los diferentes fármacos en la eliminación de

nutrientes mediante microalgas, se llevó a cabo mediante el análisis de la concentración de
nitratos (NO3‐) y fosfatos (PO43‐) por espectrofotometría UV/Vis, durante el cultivo discontinuo y
semicontinuo.
Los resultados de eliminación de nitratos y fosfatos por cada especie de microalga bajo la
presencia de paracetamol, ácido salicílico y diclofenaco se encuentran representados en las
Figuras 9.4, 9.6 y 9.8, respectivamente. Como se puede ver, estas curvas muestran una tendencia
similar a las curvas de crecimiento (Figuras 9.1‐9.3) ya que el crecimiento de las microalgas se
lleva a cabo gracias al consumo de nutrientes. Así mismo, los parámetros cinéticos de ajuste sobre
la eliminación nutrientes se recogen en la Tabla 9.2, 9.3 y 9.4 para los tratamientos con
paracetamol, ácido salicílico y diclofenaco, respectivamente.
9.2.1. Eliminación de nutrientes bajo la presencia de paracetamol
El tratamiento de paracetamol con S. obliquus presentó una mayor fase lag en el comienzo
de la eliminación de nitratos. Sin embargo, en el caso de la eliminación de fosfatos, fue el
tratamiento con C. vulgaris el que mostró el mayor retraso en la eliminación de fosfatos
(Tabla 9.2). En cuanto a la capacidad máxima de eliminación de nutrientes, el valor obtenido para
el parámetro K, no reveló diferencias significativas entre el tratamiento con C. vulgaris y
S. obliquus, tanto en el caso de la eliminación de nitratos como en la de fosfatos (Tabla 9.2,
Figura 9.4). Además, la tasa de eliminación de nitratos no mostró diferencias significativas entre
el tratamiento con C. vulgaris y S. obliquus. Sin embargo, la tasa de eliminación de fosfatos fue
superior en el caso de S. obliquus (Tabla 9.2, Figura 9.4).
La eliminación de nutrientes por C. sorokiniana en el tratamiento con paracetamol mostró

una eliminación casi completa de ambos nutrientes al final del cultivo en modo discontinuo
(Figura 9.5), al igual que ocurrió en los tratamientos con C. vulgaris y S. obliquus (Figura 9.4). Este
resultado puede explicarse debido a que el final de la fase exponencial de crecimiento estuvo
marcada por el agotamiento de nutrientes.
142


Figura 9.4 Eficiencia volumétrica en la eliminación de (a) nitratos (NO3‐) y (b) fosfatos (PO43‐) por C. vulgaris () y
S. obliquus () en los tratamientos con paracetamol. Los puntos representan los datos experimentales y las líneas
continuas los ajustes a la cinética del modelo logístico durante el cultivo discontinuo. Los experimentos se llevaron a
cabo por triplicado, mostrando las barras de error la desviación estándar.
Figura 9.5 Eficiencia volumétrica en la eliminación de nitratos (NO3‐) y fosfatos (PO43‐) por C. sorokiniana en los
tratamientos con paracetamol. Los experimentos se llevaron a cabo por triplicado, mostrando las barras de error la
desviación estándar.
Por otra parte, durante el estado estacionario del cultivo en modo semicontinuo, la
eliminación de fosfatos fue casi completa en todos los tratamientos, siendo superior al 98%. En el
caso de los nitratos, el tratamiento con S. obliquus mostró la mayor eliminación, alcanzando
valores de eficiencia mayores del 94%. Por el contrario, el tratamiento con C. vulgaris alcanzó la
menor eficiencia, con valores superiores al 76%. En el caso del tratamiento con C. sorokiniana, los
valores fueron intermedios, con eficiencias en la eliminación de nitratos superiores al 81%
(Tabla 9.2). Sin embargo, la eficiencia específica reveló que C. sorokiniana eliminó 2,3 veces más
nitratos por gramo de biomasa que C. vulgaris y 1,4 veces más que S. obliquus. Igualmente,
C sorokiniana eliminó 2,7 veces más fosfatos que C. vulgaris y 1,8 veces más que S. obliquus
(Tabla 9.4). Este resultado se puede explicar por la combinación de la eficiencia de eliminación y
143

CAPÍTULO 9
la menor concentración de biomasa alcanzada en el tratamiento con C. sorokiniana respecto al

alcanzado por las otras especies.
Tabla 9.2 Parámetros cinéticos del modelo logístico (a, K, r) determinados para la eliminación de nitratos (NO3‐) y
fosfatos (PO43‐) en los tratamientos con paracetamol en el cultivo en modo discontinuo de C. sorokiniana (PCS),
C. vulgaris (PCV) y S. obliquus (PSO). Eficiencias volumétricas y eficiencias específicas alcanzadas en el estado
estacionario del cultivo en modo semicontinuo. Nota: n=3.
PCS PCV PSO
a ‐ 3,84 ± 0,27 5,07 ± 1,09
K (mg l‐1) ‐ 749,09 ± 28,59 700,85 ± 19,19
r (d‐1) ‐ 0,71 ± 0,11 1,11 ± 0,23

NO3‐
R2 ‐ 0,9826 0,9791
Eficiencia volumétrica (mg l‐1 d‐1) 167,04 ± 12,31 178,82 ± 1,65 206,97 ± 1,57
Eficiencia específica (mg g biomasa‐1 d‐1) 142,86 ± 8,68 62,11 ±1,07 101,61 ± 1,17
a ‐ 6,52 ± 0,65 2,55 ± 0,43
K (mg l‐1) ‐ 54,34 ± 0,14 53,67 ± 0,42
r (d‐1) ‐ 1,51 ± 0,16 2,29 ± 0,60

PO43‐
R2 ‐ 0,9951 0,9823
Eficiencia específica (mg g biomasa‐1 d‐1) 13,98 ± 0,26 5,17 ± 0,09 8,01 ± 0,14
correlación.
9.2.2. Eliminación de nutrientes bajo la presencia de ácido salicílico
Al igual que ocurrió en el caso del paracetamol, el tratamiento de ácido salicílico con
S. obliquus presentó una mayor fase lag en el comienzo de la eliminación de nitratos. Sin embargo,
en el caso de la eliminación de fosfatos, fue el tratamiento con C. vulgaris el que mostró el mayor
retraso en la eliminación de fosfatos (Tabla 9.3, Figura 9.6). Así mismo, la capacidad máxima de
eliminación de nutrientes no mostró diferencias significativas entre el tratamiento de ácido
salicílico con C. vulgaris y S. obliquus. Sin embargo, la tasa de eliminación de fosfatos fue superior
en el caso de S. obliquus (Tabla 9.3, Figura 9.6), al igual que ocurrió en el caso del paracetamol.
144

La eliminación de nutrientes por C. sorokiniana en el tratamiento con ácido salicílico mostró
una eliminación casi completa de ambos nutrientes al final del cultivo discontinuo (Figura 9.7), al
igual que ocurrió en los tratamientos con C. vulgaris y S. obliquus (Figura 9.6). Como ya fue
indicado para el paracetamol, este resultado puede explicarse porque el final de la fase
exponencial de crecimiento ocurre debido al agotamiento de nutrientes.
Figura 9.6 Eficiencia volumétrica en la eliminación de (a) nitratos (NO3‐) y (b) fosfatos (PO43‐) por C. vulgaris () y
S. obliquus () en los tratamientos con ácido salicílico. Los puntos representan los datos experimentales y las líneas
continuas los ajustes a la cinética del modelo logístico durante el cultivo discontinuo. Los experimentos se llevaron a
cabo por triplicado, mostrando las barras de error la desviación estándar.
Figura 9.7 Eficiencia volumétrica en la eliminación de nitratos (NO3‐) y fosfatos (PO43‐) por C. sorokiniana en los
tratamientos con ácido salicílico. Los experimentos se llevaron a cabo por triplicado, mostrando las barras de error la
desviación estándar.
eliminación de fosfatos fue casi completa en todos los tratamientos, siendo superior al 98%. En el
caso de los nitratos, el tratamiento con S. obliquus mostró la mayor eliminación, alcanzando
valores de eficiencia mayores del 99%, en el tratamiento con C. vulgaris valores superiores al 93%
y en el caso del tratamiento con C. sorokiniana, mostró eficiencias en la eliminación de nitratos
superiores al 80% (Tabla 9.3). Sin embargo, la eficiencia específica, reveló que la eliminación de
145

CAPÍTULO 9
nutrientes por gramo de biomasa alcanzada por C. sorokiniana fue más de 4 veces superior a la
eliminación de S. obliquus y más de 2,7 veces que la eliminación de C. vulgaris (Tabla 9.3). Estas
diferencias, como ocurría en el tratamiento con paracetamol, se pueden explicar por la
combinación de la eficiencia de eliminación y la menor concentración de biomasa alcanzada por
C. sorokiniana respecto a las otras dos especies.
fosfatos (PO43‐) en los tratamientos con ácido salicílico en el cultivo discontinuo de C. sorokiniana (SCS), C. vulgaris
(SCV) y S. obliquus (SSO). Eficiencias volumétricas y eficiencias específicas alcanzadas en el estado estacionario del
cultivo en modo semicontinuo. Nota: n=3.
SCS SCV SSO
a ‐ 3,95 ± 0,66 4,73 ± 0,49
K (mg l‐1) ‐ 719,01 ± 1,75 732,48 ± 18,00
r (d‐1) ‐ 0,88 ± 0,16 0,95 ± 0,09

NO3‐
R2 ‐ 0,9736 0,9871
a ‐ 5,40 ± 0,99 3,59 ± 0,45
K (mg l‐1) ‐ 54,88 ± 0,51 54,41 ± 0,83
r (d‐1) ‐ 1,42 ± 0,26 1,99 ± 0,24

PO43‐
R2 ‐ 0,9966 0,9905
correlación.
9.2.3. Eliminación de nutrientes bajo la presencia de diclofenaco
El parámetro a reveló que los tratamientos con las especies del género Chlorella estudiadas
fueron significativamente más rápidos en el comienzo de la eliminación de nitratos que el
tratamiento con S. obliquus, no mostrando diferencias significativas entre ellas. Sin embargo,
C. sorokiniana mostró el mayor retraso en la eliminación de fosfatos, seguido de C. vulgaris
(Tabla 9.4). En cuanto a la capacidad máxima de eliminación de nitratos, no se detectaron
diferencias significativas entre las especies. Sin embargo, en el caso de los fosfatos, el tratamiento
146

con C. vulgaris mostró una capacidad significativamente menor que C. sorokiniana y S. obliquus, las
cuales no mostraron diferencias significativas entre ellas (Tabla 9.4, Figura 9.8). Además, la tasa
de eliminación de nitratos del tratamiento con C. vulgaris mostró un valor mucho menor que el
alcanzado en los otros dos tratamientos, los cuales no mostraron diferencias significativas entre
ellos. De la misma manera, el tratamiento con C. vulgaris mostró la menor tasa de eliminación de
fosfatos, mientras que la mayor fue la de C. sorokiniana (Tabla 9.4, Figura 9.8).
En cualquier caso, al final del cultivo en modo discontinuo, la eliminación de nutrientes fue
casi completa para las especies aquí consideradas, ya que, como ha sido referido anteriormente, el
final de la fase exponencial de crecimiento estuvo marcado por el agotamiento de nutrientes.
Figura 9.8 Eficiencia volumétrica en la eliminación de (a) nitratos (NO3‐) y (b) fosfatos (PO43‐) por
C. sorokiniana (●), C. vulgaris () y S. obliquus () en los tratamientos con diclofenaco. Los puntos representan los
datos experimentales y las líneas continuas los ajustes a la cinética del modelo logístico durante el cultivo
discontinuo. Los experimentos se llevaron a cabo por triplicado, mostrando las barras de error la desviación estándar.
eliminación de nitratos fue casi 1,2 veces menor que la eliminación de fosfatos para las especies
aquí estudiadas. El tratamiento con S. obliquus mostró la mayor eliminación de nutrientes,
alcanzando valores de eficiencia mayores del 87% para nitratos y 99% para fosfatos. Por el
contrario, el tratamiento con C. vulgaris alcanzó la menor eficiencia, con valores cercanos al 66%
en la eliminación de nitratos y al 78% para los fosfatos. Por su parte, C. sorokiniana logró una
eliminación de nitratos superior al 82% y de fosfatos mayor del 98%. Los resultados relativos a la
eficiencia específica, es decir, la relación entre la eficiencia volumétrica y la concentración de
biomasa, revelaron que S. obliquus eliminó cerca de 1,5 veces más nutrientes que C. sorokiniana y
cerca de 2 veces más que C. vulgaris (Tabla 9.4). En el caso del diclofenaco, fue S. obliquus la
especie que alcanzó un menor concentración de biomasa, como puede observarse en la Figura 9.3,
por lo que la relación entre eliminación respecto a biomasa fue mayor.
147

CAPÍTULO 9
fosfatos (PO43‐) en los tratamientos con diclofenaco en el cultivo discontinuo de C. sorokiniana (DCS), C. vulgaris
(DCV) y S. obliquus (DSO). Eficiencias volumétricas y eficiencias específicas alcanzadas en el estado estacionario del
DCS DCV DSO
a 2,19 ± 0,53 2,61 ± 0,14 3,24 ± 0,21
K (g l‐1) 734,80 ± 11,09 791,91 ± 16,98 758,26 ± 6,00
r (d‐1) 0,83 ± 0,15 0,56 ± 0,09 0,84 ± 0,06

NO3‐
R2 0,9585 0,9789 0,9809
a 4,18 ± 0,16 3,59 ± 0,52 2,50 ± 0,32
K (g l‐1) 55,39 ± 0,06 48,49 ± 6,59 55,60 ± 0,59
r (d‐1) 1,20 ± 0,06 0,69 ± 0,00 0,85 ± 0,02

PO43‐
R2 0,9962 0,9624 0,9683
correlación.
El hecho de que la eliminación de fosfatos sea mayor que la de nitratos durante el estado
estacionario del cultivo en modo semicontinuo puede explicarse por la relación de nitrógeno y
fósforo (N:P). Durante el cultivo semicontinuo hay una limitación de fósforo debido a una tasa de
eliminación de fosfatos más rápida y la adición diaria de medio de cultivo. Esto hace que se
produzca una acumulación de nitratos, siendo por tanto incompleta su eliminación.
A la vista de los resultados obtenidos se puede destacar que, en los tratamientos con
fármacos, tanto los nitratos como los fosfatos fueron eliminados casi por completo al final del
cultivo en modo discontinuo por las tres especies de microalgas, del mismo modo que ocurrió con
los controles positivos de cada especie. Sin embargo, durante el estado estacionario del cultivo
semicontinuo, S. obliquus alcanzó en todos los casos las mayores eficiencias de eliminación de
nutrientes, obteniendo los peores resultados con C. sorokiniana en los tratamientos con
paracetamol y ácido salicílico y con C. vulgaris en el caso del diclofenaco. Sin embargo, la
eliminación por gramo de biomasa, mostró que C. sorokiniana eliminó más nutrientes por gramo
de biomasa en los tratamientos con paracetamol y ácido salicílico, y S. obliquus en el tratamiento
148

con diclofenaco. En el caso de los controles positivos, a pesar de alcanzar en general una menor
eficiencia de eliminación de nutrientes, fue consecuencia de un menor crecimiento en los
controles que en los tratamientos. De cualquier forma, la presencia de los fármacos estudiados no
interfirió negativamente con la eliminación de nutrientes en ningún caso.
Los resultados obtenidos confirman la viabilidad de las especies estudiadas en la

eliminación de nutrientes y su aplicabilidad en los tratamientos de aguas residuales, tal y como ha
sido declarado anteriormente (Arbib et al., 2013; Cabanelas et al., 2013; Gómez et al., 2015;
Prandini et al., 2016). Sin embargo, es necesario destacar la existencia de una limitación de
nutrientes en las aguas residuales de las EDARs, con una concentración típica de nitrógeno en
forma de amonio de 20‐40 mg l‐1 y fósforo de 1‐10 mg l‐1 (McGinn et al., 2011). A pesar de esta
limitación, estos autores (Arbib et al., 2013; Cabanelas et al., 2013; Gómez et al., 2015; Prandini et
al., 2016) han demostrado que la producción de biomasa es compatible con la eliminación de
nutrientes de aguas residuales. En los últimos años se han llevado a cabo numerosos estudios en
el tratamiento del efluente procedente de la digestión anaerobia de los lodos (AlMomani and
Örmeci, 2016; Ledda et al., 2015; Morales‐Amaral et al., 2015a, 2015b; Sepúlveda et al., 2015),
donde se puede encontrar una mayor concentración de nutrientes, siendo necesario realizar
diluciones para evitar inhibiciones por altas concentraciones de amonio (Morales‐Amaral et al.,
2015b). Hasta donde sabemos, no existen en la literatura científica resultados sobre los efectos
que la presencia de fármacos tienen sobre la eliminación de nutrientes en sistemas de
biorremediación con microalgas.
9.3. ELIMINACIÓN DE FÁRMACOS DURANTE LA BIORREMEDIACIÓN DE AGUAS

CONTAMINADAS
El estudio de la eliminación de paracetamol, ácido salicílico y diclofenaco por parte de

C. sorokiniana, C. vulgaris y S. obliquus, se llevó a cabo por medio del análisis de la concentración
residual de cada fármaco en el medio de cultivo mediante análisis por cromatografía líquida, tanto
en los tratamientos con microalgas como en los controles negativos. Los ensayos se llevaron a
cabo en modo discontinuo y semicontinuo en fotobiorreactores bajo condiciones controladas.
La concentración de los fármacos estudiados en los controles negativos se mantuvo estable

a lo largo de la duración de los experimentos. Por lo tanto, se puede asumir que la disminución de
la concentración de los fármacos estudiados en los tratamientos con microalgas estuvo asociada a
la presencia de éstas.
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CAPÍTULO 9

Las Figuras 9.9, 9.10 y 9.11 representan los resultados de la eliminación de paracetamol,
ácido salicílico y diclofenaco, respectivamente, durante el tratamiento con C. sorokiniana,
C. vulgaris y S. obliquus. Así mismo, los parámetros cinéticos de ajuste sobre la eliminación de
dichos fármacos se recogen en la Tabla 9.5.
9.3.1. Eliminación de paracetamol
Como confirman los valores del parámetro a, no hubo diferencias significativas entre
C. vulgaris y S. obliquus en la fase lag para la eliminación de paracetamol. Sin embargo,
C. sorokiniana mostró un mayor retraso en el comienzo de la eliminación de este fármaco en
comparación con las otras dos especies (Tabla 9.5). Por otra parte, los valores obtenidos para el
parámetro K (Tabla 9.5), revelan que C. sorokiniana alcanzó una capacidad máxima de eliminación
de paracetamol 2,8 veces superior a C. vulgaris y 1,7 veces mayor que S. obliquus (Figura 9.9). De
la misma manera, las tasas de eliminación mostraron diferencias significativas entre los
tratamientos, siendo C. sorokiniana la que mostró la tasa de eliminación más rápida y C. vulgaris la
más lenta; lo cual está en consonancia con los valores determinados para el parámetro K
(Tabla 9.5).
Como consecuencia de las diferentes respuestas obtenidas en los parámetros cinéticos de

eliminación de cada especie, al final del cultivo en modo discontinuo, se alcanzaron eficiencias en
la eliminación de paracetamol superiores al 67% en el cultivo de C. sorokiniana, 21% en C. vulgaris
y 40% en S. obliquus. Estos resultados evidencian una mayor capacidad de eliminación de
paracetamol mediante C. sorokiniana, seguido por S. obliquus y C. vulgaris.
Durante el estado estacionario del cultivo en modo semicontinuo, se alcanzaron eficiencias

de eliminación de paracetamol superiores al 41% en el cultivo de C. sorokiniana, 12% en
C. vulgaris y 9% en S. obliquus (Figura 9.9). Además, la relación entre la eficiencia volumétrica y la
concentración de biomasa, descrita como eficiencia específica en la Tabla 9.5, reveló que
C. sorokiniana fue capaz de eliminar 7,2 veces más paracetamol por gramo de biomasa que
C. vulgaris y 8,4 veces más que S. obliquus. Por otra parte, la eficiencia específica en la eliminación
de paracetamol fue similar entre S. obliquus y C. vulgaris.
150


Figura 9.9 Eficiencias volumétricas en la eliminación de paracetamol mediante C. sorokiniana (PCS ●), C. vulgaris
(PCV ) y S. obliquus (PSO ) durante el cultivo en modo discontinuo (a). Los puntos representan los datos
experimentales y las líneas continuas los ajustes a la cinética del modelo logístico durante el cultivo en modo
discontinuo. Eficiencia volumétrica en la eliminación de paracetamol en el estado estacionario del cultivo en modo
semicontinuo (b). Los experimentos se llevaron a cabo por triplicado, mostrando las barras de error la desviación
estándar.
9.3.2. Eliminación de ácido salicílico
En el caso de la eliminación de ácido salicílico, C. sorokiniana mostró un retraso significativo

en el comienzo de la eliminación del fármaco en comparación con la fase lag de C. vulgaris y
S. obliquus, como muestran los valores del parámetro a en la Tabla 9.5. Los resultados obtenidos
para la máxima capacidad de eliminación (parámetro K) revelaron que S. obliquus eliminó 1,4
veces más ácido salicílico que C. sorokiniana y 3,8 veces más que C. vulgaris. Por lo tanto, al final
del cultivo en modo discontinuo, se alcanzó una eficiencia de eliminación superior al 73% en el
cultivo con C. sorokiniana, 25% con C. vulgaris y 93% con S. obliquus, a pesar de que la tasa de
eliminación de ácido salicílico alcanzada por S. obliquus fue 5.4 veces menor que la alcanzada por
C. sorokiniana (Figura 9.10).
Por otra parte, las eficiencias volumétricas medias alcanzadas en el estado estacionario del
cultivo en modo semicontinuo, no mostraron diferencias significativas entre las especies
C. sorokiniana (> 93%) y S. obliquus (> 99%) (Figura 9.10). Sin embargo, la eficiencia volumétrica
alcanzada por C. vulgaris (> 22%) fue 4 veces menor que la alcanzada por las otras dos especies.
Además, la eficiencia específica obtenida reveló que C. sorokiniana eliminó más de 12,4 veces más
ácido salicílico que C. vulgaris y 4,5 veces más que S. obliquus por gramo de biomasa (Tabla 9.5).
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CAPÍTULO 9

Figura 9.10 Eficiencias volumétricas en la eliminación de ácido salicílico mediante C. sorokiniana (SCS ●),
C. vulgaris (SCV ) y S. obliquus (SSO ) durante el cultivo discontinuo (a). Los puntos representan los datos
discontinuo. Eficiencia volumétrica en la eliminación de ácido salicílico en el estado estacionario del cultivo en modo
semicontinuo (b). Los experimentos se llevaron a cabo por triplicado, mostrando las barras de error la desviación
estándar.
9.3.3. Eliminación de diclofenaco
En la eliminación de diclofenaco durante el cultivo en modo discontinuo, las tres especies

mostraron la misma respuesta respecto al retraso en la eliminación del fármaco, por lo que se
obtuvieron valores similares en el parámetro a (Tabla 9.5) para todos los tratamientos. Sin
embargo, la capacidad máxima de eliminación mostró diferencias significativas en el tratamiento
de S. obliquus, eliminando 1,5 veces más diclofenaco que C. sorokiniana y 1,4 veces más que
C. vulgaris (Figura 9.11). Respecto a la tasa de eliminación, los resultados obtenidos revelaron
diferencias significativas entre los tratamientos. La tasa de eliminación más rápida fue alcanzada
por C. sorokiniana, alcanzando valores 1,6 veces mayores que S. obliquus y 1,4 veces mayores que
C. vulgaris. A pesar de las diferencias observadas en los parámetros de eliminación, al final del
cultivo en modo discontinuo, se alcanzaron eficiencias de eliminación superiores al 65% en el
cultivo de C. sorokiniana, 69% por C. vulgaris y 98% por S. obliquus.
Durante el estado estacionario del cultivo en modo semicontinuo, el tratamiento con

S. obliquus alcanzó eficiencias de eliminación de diclofenaco (> 79%) 2,6 veces superiores a las
alcanzadas por C. sorokiniana y 3,7 veces superiores que C. vulgaris (Figura 9.11). Además, los
valores determinados para la eficiencia específica revelaron que S. obliquus eliminó 3 veces más
diclofenaco por gramo de biomasa que C. sorokiniana y 5,4 veces más que C. vulgaris (Tabla 9.5).
152


Figura 9.11 Eficiencias volumétricas en la eliminación de diclofenaco mediante C. sorokiniana (DCS ●), C. vulgaris
(DCV ) y S. obliquus (DSO ) durante el cultivo discontinuo (a). Los puntos representan los datos experimentales y
las líneas continuas los ajustes a la cinética del modelo logístico durante el cultivo en modo discontinuo. Eficiencia
volumétrica en la eliminación de diclofenaco en el estado estacionario del cultivo en modo semicontinuo (b). Los
experimentos se llevaron a cabo por triplicado, mostrando las barras de error la desviación estándar.
El hecho de que las curvas de eliminación muestren una tendencia similar a las curvas de
crecimiento indica una relación entre el crecimiento de las microalgas y la eficiencia de
eliminación de los fármacos. A la vista de los resultados obtenidos, se puede concluir que el
paracetamol fue eliminado con una mayor eficiencia por C. sorokiniana, tanto por litro como por
gramo de biomasa (> 67% en el cultivo en modo discontinuo, > 41% en el cultivo en modo
semicontinuo) a pesar de que la concentración de biomasa alcanzada en el cultivo fue la menor de
las tres especies. Además, la tasa de eliminación de C. sorokiniana fue la más rápida incluso
contando con la menor tasa de crecimiento entre los tratamientos con paracetamol. Sin embargo,
la presencia de paracetamol en el cultivo de C. sorokiniana provocó el mayor incremento en la
concentración de biomasa respecto al control positivo (> 49%).
Por otra parte, S. obliquus mostró la mayor capacidad de eliminación de ácido salicílico
durante el cultivo en modo discontinuo (> 93%) así como durante el estado estacionario del
cultivo semicontinuo (> 99%). Sin embargo, la tasa de eliminación de ácido salicílico de esta
especie fue la menor de entre las aquí estudiadas. La mayor tasa de eliminación fue alcanzada por
el cultivo de C. sorokiniana, mostrando una eliminación de ácido salicílico por gramo de biomasa
4,5 veces mayor que S. obliquus. Además, el incremento de la biomasa por la presencia de ácido
salicílico fue superior al 52% en el tratamiento con C. sorokiniana, mientras que para S. obliquus
fue superior al 36%.
Respecto a la eliminación de diclofenaco, a pesar de que las células de C. sorokiniana

lograron una mayor tasa eliminación y una mayor tasa de crecimiento, S. obliquus fue la especie
que alcanzó la mayor eficiencia de eliminación de diclofenaco (> 98% en el cultivo en modo
153

CAPÍTULO 9

discontinuo, > 79% en el cultivo en modo semicontinuo), tanto por litro como por gramo de
biomasa.
Comparando los tres fármacos, el ácido salicílico fue el fármaco eliminado con una mayor
eficiencia, mostrando C. sorokiniana y S. obliquus los mejores resultados. Contrariamente, el
paracetamol fue el fármaco que se eliminó con una peor eficiencia. En todos los casos, C. vulgaris
mostró las eficiencias más bajas para los tres fármacos. Estos resultados pueden estar
relacionados con las características específicas de cada especie, los mecanismos involucrados en
la eliminación y con las propiedades de cada fármaco.
Los resultados publicados sobre eliminación de contaminantes emergentes por microalgas

revelan, como ocurre en este trabajo, diferentes eficiencias en función del contaminante y de la
especie de microalga. Por ejemplo, Gattullo et al. (2012) demostraron que la microalga
Monoraphidium braunii fue capaz de eliminar más del 48% de bisfenol A, partiendo de una
concentración inicial de 4 mg l‐1. De Wilt et al. (2016) alcanzaron eficiencias superiores al
60‐100% para la eliminación de diclofenaco, ibuprofeno, paracetamol y metoprolol mediante
C. sorokiniana cultivada en aguas residuales. Sin embargo, ante idénticas condiciones, la
eliminación de carbamazepina y trimetoprima fue incompleta y no excedió de 30 y 60%,
respectivamente (de Wilt et al., 2016). Wang et al, (2016) estudiaron la eliminación de fenol por
Chlorella sp., obteniendo eficiencias de eliminación del 100% en siete días, partiendo de una
concentración inicial de 500 mg l‐1. Peng et al. (2014) determinaron eliminaciones de
progesterona superiores al 95% mediante S. obliquus y Chlorella pyrenoidosa. En el caso de
norgestrel, las eliminaciones logradas por S. obliquus fueron casi completas y al menos del 40%
por C. pyrenoidosa (Peng et al., 2014). Igualmente, Hom‐Díaz et al. (2015) estudiaron la
eliminación de las hormonas E2 y EE2 del efluente procedente de la digestión anaerobia de los
lodos de una EDAR mediante las microalgas Selenastrum capricornutum y Chlamydomonas
reinhardtii. Tras siete días de cultivo, estos autores (Hom‐Díaz et al., 2015) lograron eliminaciones
superiores al 88% para E2 y superiores al 60% para EE2. Además, Matamoros et al. (2015)
estudiaron la capacidad de los sistemas de tratamiento de aguas residuales basados en microalgas
para eliminar 25 contaminantes orgánicos. En el caso del diclofenaco lograron eficiencias entre
82‐92% en función del tiempo de retención.
154


Tabla 9.5 Parámetros cinéticos del modelo logístico (a, K, r) determinados para la eliminación de paracetamol, ácido
salicílico y diclofenaco en el cultivo discontinuo de C. sorokiniana (PCS, SCS, DCS), C. vulgaris (PCV, SCV, DCV) y
S. obliquus (PSO, SSO, DSO). Eficiencias volumétricas y eficiencias específicas alcanzadas en el estado estacionario del
Paracetamol PCS PCV PSO
a 4,49 ± 0,24 3,84 ± 0,01 3,19 ± 0,58
K (mg l‐1) 17,62 ± 0,91 6,23 ± 0,02 10,41 ± 1,58
r (d‐1) 1,01 ± 0,06 0,77 ± 0,01 0,86 ± 0,21
R2 0,9941 0,9827 0,9766
Ácido salicílico SCS SCV SSO
a 10,20 ± 3,16 4,09 ± 0,87 4,11 ± 0,16
K (mg l‐1) 17,68 ± 0,96 6,44 ± 0,63 24,67 ± 0,32
r (d‐1) 4,07 ± 1,21 0,84 ± 0,17 0,76 ± 0,03
R2 0,9919 0,9947 0,9973
Diclofenaco DCS DCV DSO
a 3,88 ± 0,62 3,23 ± 0,02 3,01 ± 0,38
K (mg l‐1) 14,55 ± 0,73 15,52 ± 0,26 22,43 ± 0,20
r (d‐1) 2,03 ± 0,33 1,44 ± 0,05 1,25 ± 0,19
R2 0,9626 0,9755 0,9690
correlación.Eliminación de altas concentraciones relativas de paracetamol y ácido salicílico
155

CAPÍTULO 9

9.3.4. Eliminación de altas concentraciones relativas de paracetamol y ácido salicílico
En la Figura 9.12 se representan los resultados de eliminación de paracetamol y ácido

salicílico por C. sorokiniana para concentraciones iniciales un orden de magnitud superior a las
anteriormente consideradas, mientras que los parámetros de ajuste correspondientes se
encuentran en la Tabla 9.6.
Figura 9.12 Eficiencias volumétricas en la eliminación de altas concentraciones relativas de paracetamol (●) y ácido
salicílico () durante el cultivo de C. sorokiniana en modo discontinuo (a). Los puntos representan los datos
discontinuo. Eficiencia volumétrica en la eliminación de altas concentraciones relativas de paracetamol y ácido
salicílico en el estado estacionario del cultivo en modo semicontinuo (b). Los experimentos se llevaron a cabo por
triplicado, mostrando las barras de error la desviación estándar.
Como puede verse, la concentración de 250 mg l‐1 de paracetamol no modificó el tiempo de

retardo en el comienzo de la eliminación mediante C. sorokiniana (Tabla 9.6) en comparación con
el valor obtenido por la adición de 25 mg l‐1 (Tabla 9.5). Sin embargo, en el caso del ácido
salicílico, la mayor concentración de fármaco (250 mg l‐1) provocó un aumento significativo de la
fase lag en comparación con el valor obtenido para 25 mg l‐1, lo que está relacionado con el
retraso en el comienzo de la fase exponencial de crecimiento. Por otra parte, las tasas de
eliminación de ambos fármacos no se vieron modificadas por el incremento de la concentración
inicial de fármaco (Tabla 9.6). Tanto para 25 mg l‐1 como para 250 mg l‐1, la capacidad máxima de
eliminación del ácido salicílico se alcanzó en 4 días, mientras que fueron necesarios 7‐8 días para
el paracetamol, siendo el valor de este parámetro de un orden de magnitud superior al alcanzado
con la concentración inicial de 25 mg l‐1. Además, la tasa de eliminación del ácido salicílico fue de
4 (concentración inicial de 25 mg l‐1) a 5 (concentración inicial de 250 mg l‐1) veces superior a la
del paracetamol. Al final del cultivo en modo discontinuo, se alcanzaron eficiencias superiores al
94% en el caso del ácido salicílico y superiores al 48% en el paracetamol (Figura 9.12), en
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comparación con el 73% de ácido salicílico y 67% de paracetamol eliminados para
concentraciones iniciales de 25 mg l‐1 (Figuras 9.9‐9.10).
Tabla 9.6 Parámetros cinéticos del modelo logístico (a, K, r) determinados para la eliminación de altas
concentraciones relativas de paracetamol y ácido salicílico en el cultivo discontinuo de C. sorokiniana. Nota: n=3.
Paracetamol Ácido salicílico
a 3,98 ± 1,27 14,90 ± 0,05
K (mg l‐1) 146,46 ± 41,01 234,38 ± 0,69
r (d‐1) 0,90 ± 0,45 4,57 ± 0,07
R2 0,9657 0,9990
correlación.
No tenemos constancia de estudios previos sobre la eliminación de fármacos a diferentes

concentraciones mediante microalgas y los datos existentes sobre la eliminación mediante otros
microorganismos es escasa. La biodegradación bacteriana de paracetamol a diferentes
concentraciones ha sido estudiada por diversos autores (Gusseme et al., 2011; Wu et al., 2012;
Zhang et al., 2013), logrando degradaciones altas o completas incluso a concentraciones iniciales
de 2.500 mg l‐1 por especies del género Pseudomonas (Zhang et al., 2013). Por otra parte,
Combarros et al. (2014) observaron una biodegradación de ácido salicílico por Pseudomonas
putida del 100% y 94.7% a concentraciones iniciales de 100 y 500 mg l‐1, respectivamente. Por lo
tanto, aunque en un rango diferente de concentraciones, estos autores (Combarros et al., 2014)
verificaron la disminución de la eficiencia al incrementar la concentración inicial del fármaco,
contrariamente a lo observado en nuestros estudios.
De forma global, los resultados obtenidos en cuanto a la eliminación de fármacos mediante

microalgas indican la viabilidad de la utilización de las microalgas aquí estudiadas como sistema
de biorremediación. Además ha quedado demostrada no solo la resistencia, sino también la
capacidad de eliminación de altas concentraciones relativas de paracetamol y ácido salicílico
mediante C. sorokiniana. Estos resultados apuntan al potencial uso de las microalgas en la
biorremediación del efluente procedente de la digestión anaerobia de lodos, donde los
contaminantes se encuentran en una gran concentración. Además de las diferentes eficiencias en
función del fármaco y la especie de microalga, estos resultados evidencian que la concentración
del fármaco influye en la eficiencia alcanzada. En cualquier caso, aún es necesaria una mayor
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investigación para evaluar los mecanismos de eliminación involucrados en la eliminación

fármacos por parte de las diferentes especies de microalgas.
9.4. COAGULACIÓN‐FLOAGULACIÓN COMO SISTEMA DE SEPARACIÓN DE LA BIOMASA

DE MICROALGAS DEL AGUA TRATADA
Los ensayos de separación de la biomasa mediante coagulación‐floculación se llevaron a

cabo con la biomasa cosechada al final del cultivo discontinuo y del estado estacionario del cultivo
semicontinuo. La concentración de la biomasa se midió por densidad óptica mediante
espectrofotometría visible, tomando muestras de la zona clarificada a los 0, 1, 5, 10, 15, 30 y 60
minutos tras la adición del floculante. Además, los ensayos de coagulación‐floculación se llevaron
a cabo con la biomasa procedente de los tratamientos con fármacos, verificándose que la
presencia de fármacos no afectó a la eficiencia de los floculantes en los casos aquí estudiados.
La sedimentación natural de las especies del género Chlorella aquí estudiadas fue
prácticamente inexistente, incluso con tiempos de incubación superiores a los 60 minutos. Esto se
debe relacionar con la combinación del pequeño celular (3‐5 µm), la baja concentración en el
medio de cultivo (0,5‐2,5 g l‐1) y las fuerzas electrostáticas de repulsión entre las células (Grima et
al., 2003). Por todo ello, la velocidad de sedimentación natural de Chlorella es menor de 10‐6 m s‐1,
lo que hace que no sea un sistema eficiente (Granados et al., 2012). Sin embargo, la capacidad de
floculación natural de S. obliquus (5‐10 µm) fue mayor, alcanzando eficiencias de eliminación
mayores del 26% en 60 minutos y logrando eficiencias próximas al 100% tras 16 horas. Aparte
del tamaño celular, las diferencias en la sedimentación natural de las especies del género Chlorella
aquí estudiadas y S. obliquus también pueden estar relacionadas con la composición de las
sustancias poliméricas hidrofóbicas extracelulares de las células, las cuales constituyen un
importante factor en la sedimentación natural de las microalgas (Salim et al., 2014).
Los eficiencias obtenidas con los diferentes floculantes estudiados con la biomasa de
C. sorokiniana, C. vulgaris y S. obliquus, cosechada al final del cultivo discontinuo, se representan
en la Figura 9.13 para el caso de las sales metálicas, en la Figura 9.14 para los polielectrolitos y en
la Figura 9.15 para el chitosan.
9.4.1. Coagulación‐floculación mediante sales metálicas
En el caso de C. sorokiniana, las sales de AlCl3 presentaron mayores eficiencias que las de
FeCl3 para la misma dosis, con la biomasa cosechada al final del cultivo en modo discontinuo. Se
alcanzaron eficiencias superiores al 95% tras 1 minuto de incubación, tal y como muestra la
158

Figura 9.13. Las dosis que resultaron más eficientes fueron 200 mg g‐1 para AlCl3 (> 95%, 1 min) y
250 mg g‐1 para FeCl3 (> 98%, 1 min). En todos los casos, tiempos de incubación mayores de 15
minutos supusieron incrementos de la eficiencia menores del 5%.
Los resultados de coagulación‐floculación con C. vulgaris mostraron que el AlCl3

(Figura 9.13) presentó mayores eficiencias a bajas concentraciones que el FeCl3 (Figura 9.13), no
mostrando grandes diferencias entre las dosis testadas. Sin embargo, los resultados obtenidos con
las mayores dosis (200, 250, 300 mg g‐1), mostraron que las sales de hierro lograron mayores
eficiencias en un tiempo ligeramente menor que las sales de aluminio. Las dosis que resultaron
más eficientes fueron 250 mg g‐1 para AlCl3 (> 95%, 5 min) y 200 mg g‐1 para FeCl3
(> 95%, 1 min). Tiempos de incubación mayores de 15 minutos supusieron un incremento de la
eficiencia inferior al 5%, excepto para las dosis más bajas de FeCl3 (100 y 150 mg g‐1), que
supusieron un aumento del 17%.
Por otra parte, S. obliquus mostró eficiencias con AlCl3 claramente más bajas que las
obtenidas con FeCl3 (Figura 9.13), obteniendo recuperaciones de la biomasa más bajas con ambas
sales que las logradas para C. sorokiniana y C. vulgaris. De hecho, fue necesario utilizar la dosis
más alta testada (300 mg g‐1) y tiempos de incubación de 60 minutos para lograr eficiencias del
88% utilizando AlCl3 y 99% con FeCl3. Sin embargo, en este caso, aumentar los tiempos de
incubación por encima de los 15 minutos supuso un incremento de la recuperación de la biomasa
de hasta el 35% para AlCl3 y de hasta el 20 % para FeCl3. Este incremento de la eficiencia tras 15
minutos podría explicarse por el efecto añadido de la floculación natural.
Por otra parte, las eficiencias alcanzadas con la biomasa cosechada en el estado
estacionario del cultivo en modo semicontinuo fueron menores (datos no mostrados) que los
valores alcanzados con la misma dosis en el cultivo en modo discontinuo. En el caso de
C. sorokiniana, las sales de aluminio proporcionaron una recuperación del 88% con 200 mg g‐1 y
las sales de hierro del 86% con 250 mg g‐1, con tiempos de incubación de 60 minutos. En el caso
de C. vulgaris, se obtuvieron eficiencias del 83% con 250 mg g‐1 de AlCl3 y del 87% con 200 mg g‐1
de FeCl3, con tiempos de incubación de 60 minutos. En el caso de S. obliquus, se obtuvieron
eficiencias del 78% con 300 mg g‐1 de AlCl3 y del 84% con 300 mg g‐1 de FeCl3, con tiempos de
incubación de 60 minutos.
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Figura 9.13 Eficiencias de recuperación de la biomasa al final del cultivo en discontinuo utilizando diferentes dosis
de AlCl3 en la recuperación de C. sorokiniana (a), C. vulgaris (b) y S. obliquus (c); y utilizando diferentes dosis de
FeCl3 en la recuperación de C. sorokiniana (d), C. vulgaris (e) y S. obliquus (f). Las dosis se expresan como miligramos
de floculante por gramo de biomasa. Los experimentos se llevaron a cabo por triplicado, mostrando las barras de
error la desviación estándar.
A la vista de los resultados obtenidos se puede concluir que la edad del cultivo afecta a la
eficiencia de floculación. Las diferencias en los valores de recuperación de la biomasa se podría
explicar por la dependencia de la composición de la pared celular, la edad del cultivo, el grado y
tipo de excreciones, condiciones fisiológicas y otros factores (Avnimelech et al., 1982). Además, es
sabido que la composición bioquímica de la superficie de la célula varía entre la fase exponencial
del cultivo y el estado estacionario, resultando en diferentes comportamientos en la floculación
(Danquah et al., 2009; Henderson et al., 2008). Además, las microalgas excretan materia orgánica,
160

como polisacáridos, y proteínas que se acumulan en el medio de cultivo y pueden competir con las
microalgas por los floculantes (Chen et al., 2009; X. Zhang et al., 2012).
La coagulación‐floculación con sales metálicas (Zn2+, Al3+ y Fe3+) ha sido ampliamente

estudiada, presentando mejores tasas de recuperación las sales de aluminio que las de hierro en el
caso de especies del género Chlorella (Papazi et al., 2010), concordando con los resultados
obtenidos aquí con C. sorokiniana y C. vulgaris. Sin embargo, en el caso de S. obliquus las
eficiencias alcanzadas con las sales de hierro fueron superiores a las alcanzadas con las sales de
aluminio, al igual que los resultados obtenidos por Selesu et al. (2016), quienes obtuvieron
recuperaciones de Scenedesmus sp. del 96,5 ± 1,1% con 300 mg l‐1 de Al2(SO4)3 y 96,0 ± 0,8% con
FeCl3 con 150 mg l‐1, con una concentración de la biomasa de 0,5 g l‐1 y tiempos de incubación de
30 minutos. Teniendo en cuenta la concentración de la biomasa, las recuperaciones obtenidas por
Selesu et al. (2016) con las sales de hierro fueron ligeramente superiores a las aquí obtenidas. Sin
embargo, en el caso de las sales de aluminio las eficiencias fueron mucho menores debido a ser
necesarias altas dosis y largos periodos de incubación. Por otra parte, las sales de hierro en alta
concentración alteraron el color de las células y dieron un color amarillento al sobrenadante,
como ya habían observado Papazi et al. (2010). Los estudios publicados con sales metálicas
muestran en su mayoría eficiencias menores que las aquí alcanzadas, tanto en el cultivo
discontinuo como en el semicontinuo. Papazi et al. (2010) obtuvieron eficiencias menores del
90% en la recuperación de Chlorella minutissima, cosechada de un cultivo en modo discontinuo,
con 500 mg l‐1 de AlCl3 y FeCl3 con un tiempo de sedimentación de 200 minutos y una
concentración de la biomasa de 2,20.108 células ml‐1. Rakesh et al. (2014) alcanzaron eficiencias
menores del 70% en la recuperación de C. sorokiniana de un cultivo en modo discontinuo de
2 semanas, con 200 mg l‐1 de sulfato de aluminio y menores de 85% con 32,44 mg l‐1 de cloruro
férrico. Por su parte, Granados et al. (2012) obtuvieron eficiencias de recuperación de
Muriellopsis sp. inferiores al 30 % con 20 mg l‐1 de cloruro de hierro y menores del 15% con
70 mg l‐1 de sulfato de aluminio, tras 15 minutos de incubación y de un cultivo semicontinuo con
una concentración de biomasa de 2,0 g l‐1.
9.4.2. Coagulación‐floculación mediante polielectrolitos sintéticos
Los resultados obtenidos en el caso de C. sorokiniana, el polímero catiónico CH‐35 mostró

una mayor eficiencia que el polímero catiónico CH‐15 (Figura 9.14). Con estos polielectrolitos se
alcanzaron eficiencias superiores al 99,5% con 15 mg g‐1 de CH‐35 y mayores del 98% con 20 mg
g‐1 de CH‐15 en tan solo 1 minuto de incubación. De la misma manera que ocurrió con las sales
metálicas, tiempos de incubación superiores a 15 minutos supusieron un incremento de la
recuperación del 1%.
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Las eficiencias obtenidas en el caso de C. vulgaris (Figura 9.14) mostraron resultados

similares con ambos polielectrolitos. Se obtuvieron eficiencias mayores el 99,5% con 10 mg g‐1 de
CH‐35 y CH‐15 en tan solo 1 minuto de incubación, siendo estas eficiencias mayores que las
obtenidas en el caso de C. sorokiniana. Los tiempos de incubación mayores de 15 minutos solo
supusieron un incremento del 1% en la recuperación de la biomasa, y del 10 % en la menor dosis.
En el caso de S. obliquus, el polímero catiónico (CH‐35) mostró eficiencias ligeramente

superiores que en el caso del polímero catiónico (CH‐15) (Figura 9.14), a pesar de que ambos
proporcionaron eficiencias menores que en el caso de las especies del género Chlorella aquí
estudiadas. Ninguna de la dosis testadas permitió alcanzar eficiencias mayores del 55% para el
polielectrolito CH‐35 y del 49% para el polielectrolito CH‐15, incluso con tiempos de incubación
de 15 minutos. Sin embargo, se observó que tiempos de incubación mayores de 15 minutos
proporcionaron un incremento en la recuperación de la biomasa hasta del 17% para CH‐35 y
hasta del 23% para CH‐15. Por lo tanto, para el caso de los polielectrolitos fueron necesarios
tiempos de incubación de 60 minutos y la mayor dosis testada (20 mg g‐1) para lograr
recuperaciones de biomasa del 74% con CH‐35 y del 70% con CH‐15.
En el caso de las especies del género Chlorella utilizadas en este trabajo, el efecto de los
polielectrolitos considerados tras su adición fue muy rápido pero la recuperación de biomasa se
incrementó muy poco para tiempos mayores a 15 minutos. Esto puede explicarse por el hecho de
que, en los casos en que se obtuvieron altas eficiencias relativas, estas se debieron a la generación
de un único gran flóculo inmediatamente después de adicionar el coagulante. Sin embargo, en el
caso de S. obliquus, el incremento de los tiempos de incubación supuso un incremento de las
eficiencias de recuperación de biomasa. Para esta especie, las eficiencias logradas en los primeros
minutos fueron debidas a la adición del coagulante y, posteriormente, a la capacidad natural de
sedimentación de las células.
Por otra parte, al igual que en el caso de las sales metálicas, las eficiencias alcanzadas con la
biomasa cosechada en el estado estacionario del cultivo en modo semicontinuo fueron menores
(datos no mostrados) que los valores alcanzados con la misma dosis en el cultivo en modo
discontinuo. En el caso de C. sorokiniana se lograron eficiencias superiores al 61% con 15 mg g‐1
de CH‐35 y mayores del 85% con 25 mg g‐1 de CH‐15. En el caso de C. vulgaris las eficiencias se
redujeron hasta un 74% con el polielectrolito CH‐35 y un 67% con el CH‐15, siendo necesarias
dosis de 10 mg g‐1 y 1 minuto de incubación. En el caso de S. obliquus fueron necesarias dosis y
tiempos de incubación mayores (20 mg g‐1, 60 minutos) para lograr eficiencias del 57% con CH‐35
y 52% con CH‐15.
162
Figura 9.14 Eficiencias de recuperación de la biomasa al final del cultivo en modo discontinuo utilizando diferentes
dosis de CH‐35 en la recuperación de C. sorokiniana (a), C. vulgaris (b) y S. obliquus (c); y utilizando diferentes dosis
de CH‐15 en la recuperación de C. sorokiniana (d), C. vulgaris (e) y S. obliquus (f). Las dosis se expresan como
miligramos de floculante por gramo de biomasa. Los experimentos se llevaron a cabo por triplicado, mostrando las
barras de error la desviación estándar.
Dadas las dosis relativamente bajas utilizadas con los polielectrolitos sintéticos, resultan
ser comparativamente más rápidos y eficientes que las sales metálicas en el proceso de
floculación. Sin embargo, una posible limitación del uso de polielectrolitos es la viscosidad de los
flóculos, que causó que los agregados de células se adhirieran a la pared de los tubos de ensayo
donde se realizaron los experimentos. Esta viscosidad puede, probablemente, dificultar la
recuperación de la biomasa y su posterior utilización.
A pesar de que el uso de polímeros catiónicos en la floculación de microalgas ha sido

previamente estudiado, la dosis óptima depende de la naturaleza del polímero. Así, Granados et al.
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CAPÍTULO 9
(2012) obtuvieron eficiencias mayores del 90% en la recuperación de Muriellopsis sp. procedente
de un cultivo semicontinuo, con tan solo 10 mg l‐1 de un polímero catiónico (EM 16), caracterizado
por una densidad de carga media y un peso molecular medio‐grande (concentración de la
biomasa de 2,0 g l‐1, 15 minutos de tiempo de incubación).
9.4.3. Coagulación‐floculación mediante chitosan
La recuperación de la biomasa cosechada al final del cultivo en modo discontinuo mediante

la adición de chitosan mostró eficiencias bajas incluso tras 60 minutos de incubación, siendo estos
valores alrededor del 15% en el caso de C. sorokiniana, del 53% en C. vulgaris y del 36% en
S. obliquus (Figura 9.15). Estos resultados están en consonancia con las bajas recuperaciones
obtenidas por otros investigadores (Granados et al., 2012).
Como ocurrió en el caso de las sales metálicas y los polielectrolitos sintéticos, las eficiencias
alcanzadas con chitosan con la biomasa cosechada en el estado estacionario del cultivo en modo
semicontinuo (datos no mostrados) fueron menores que las logradas con la biomasa del cultivo
discontinuo. Los valores obtenidos fueron del 39% para C. vulgaris y 30% para S. obliquus (con
200 mg g‐1 de chitosan, 60 minutos de tiempo de incubación). En el caso de C. sorokiniana se
llevaron a cabo ensayos que combinaron el chitosan con pH alcalino con el objetivo de mejorar su
eficiencia. Los mejores resultados fueron los obtenidos con una dosis de 200 mg g‐1 y un pH 10
inducido por la adición de NaOH, que permitieron una recuperación de biomasa del 69% en
15 minutos de incubación.
La combinación de chitosan y pH alcalino proporcionó mejores resultados que ambos por

separado. La falta de eficiencia del pH alcalino en la floculación obtenida en esta tesis está de
acuerdo con los resultados obtenidos por Rakesh et al. (2014), afirmando que el efecto de pH
alcalinos de valores 10‐11 no resultaron efectivos sobre la especie Chlorella sorokiniana. Sin
embargo, otros autores observaron que valores de pH entre 8,5 y 10 permitían una recuperación
superior al 90% de la microalga Phaeodactylum tricornutum (Sirin et al., 2012) o de Anabaena
marina (López et al., 2009). Las bajas eficiencias aquí obtenidas mediante pH alcalino pueden
explicarse a que esta floculación está asociada a la formación de precipitados de calcio y
magnesio, los cuales pueden tener cargas superficiales positivas e inducir la floculación por
neutralización de cargas. Por ejemplo para formar precipitados de fosfato cálcico es necesario un
exceso de iones de calcio y altas concentraciones de fosfato (Vandamme et al., 2013). Sin embargo,
en la presente tesis, debido a la eliminación de nutrientes asociada al crecimiento de las
microalgas, el nivel de nutrientes al final del cultivo en modo discontinuo y en el estado
164
estacionario del modo semicontinuo, fue baja. De hecho, la concentración de fosfatos fue reducida
en al menos un 90% en el momento en que los ensayos de floculación se llevaron a cabo.
Figura 9.15 Eficiencias de recuperación de la biomasa al final del cultivo en modo discontinuo utilizando diferentes
dosis de chitosan en la recuperación de C. sorokiniana (a), C. vulgaris (b) y S. obliquus (c). Las dosis se expresan como
miligramos de floculante por gramo de biomasa. Los experimentos se llevaron a cabo por triplicado, mostrando las
barras de error la desviación estándar.
Además del precio del floculante, en función del destino de la biomasa microalgal y del
medio de cultivo, se debe tener en cuenta la potencial contaminación causada por los floculantes
como un factor clave en su selección ya que puede limitar el posterior uso de la biomasa. A pesar
de que los floculantes inorgánicos como los cloruros de aluminio y hierro son eficientes, se
necesitan en dosis relativamente altas y pueden causar contaminación de la biomasa o del
sobrenadante con aluminio o hierro debido al exceso de iones suministrados (Granados et al.,
2012). Sin embargo, pueden ser útiles en los tratamientos de aguas residuales ya que el uso de la
biomasa es un objetivo secundario y el exceso de iones del agua tratada pueden ser eliminados
mediante columnas de intercambio iónico (Rakesh et al., 2014). Por otra parte, aunque los
polielectrolitos suponen un mayor coste, además de ser muy eficientes, no son persistentes ni
bioacumulativos y son biodegradables. En cuanto al chitosan, a pesar de no presentar efectos
tóxicos, son necesarias altas dosis para lograr eficiencias medias. Además de las distintas
eficiencias de floculación, hay que tener en cuenta que las características del flóculo formado
fueron diferentes según el coagulante‐floculante empleado. Estas características pueden limitar el
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posterior uso de la biomasa y por lo tanto, ser un factor determinante en la elección del
coagulante‐floculante.
9.5. EFECTOS SOBRE EL DESARROLLO EMBRIONARIO DEL PEZ CEBRA (DANIO RERIO)
PARA EVALUAR LA TOXICIDAD DE LOS EFLUENTES
El estudio de la disminución de la toxicidad tras la biorremediación con microalgas de

aguas contaminadas con fármacos, se llevó a cabo sobre los efluentes obtenidos tras el cultivo en
modo discontinuo, cuyas concentraciones residuales se muestran en la Figura 9.16. El organismo
modelo utilizado fue el pez cebra, observándose los efectos producidos sobre el desarrollo
embrionario hasta las 144 horas post‐fertilización (hpf). Dichos efectos se compararon con los
efectos producidos por la dosis inicial del fármaco parental. Para evitar los efectos producidos por
la composición del medio de cultivo de las microalgas, las muestras de los efluentes se diluyeron
con agua al 50, 25 o 10% en función de la eficiencia lograda por el tratamiento correspondiente.
Por lo tanto, para evaluar la disminución de la toxicidad, los efectos producidos por los efluentes
se compararon con los efectos producidos por la concentración de 12,5 mg l‐1 del correspondiente
fármaco parental. En todos los ensayos, el grupo control mostró una mortalidad igual o inferior al
10%, lo que indicó que los ensayos se llevaron a cabo en las condiciones adecuadas.
Los resultados obtenidos para la mortalidad de los embriones del pez cebra a las 80 hpf, se
muestran en las Figuras 9.17, 9.18 y 9.19 para la exposición a paracetamol, ácido salicílico y
diclofenaco, respectivamente, tanto para las soluciones experimentales del fármaco como para los
efluentes procedentes de la biorremediación con microalgas.
166
Figura 9.16 Concentración residual de fármacos durante la biorremediación de paracetamol (a), ácido salicílico (b)
y diclofenaco (c) por las microalgas C. sorokiniana (●), C. vulgaris () y S. obliquus (). Los experimentos se llevaron
a cabo por triplicado, mostrando las barras de error la desviación estándar.
9.5.1. Bioensayos con paracetamol
Los resultados obtenidos con las soluciones experimentales de paracetamol no mostraron

una incidencia significativa sobre la mortalidad en ningún tiempo de observación, tal y como se
puede ver en la Figura 9.17. Sin embargo, las anomalías (Tabla 3, Capítulo 8), que se limitaron a la
falta de pigmentación, comenzaron a observarse a las 32 hpf en los embriones expuestos a
concentraciones iguales o superiores a 2.500 µg l‐1, variando la incidencia entre 9% y 47%. A las
80 hpf el porcentaje de anomalías incrementó, observándose un exceso de pigmentación.
Sin embargo, la incidencia de anomalías aumentó drásticamente a las 144 hpf. A este tiempo: (i) el
exceso de pigmentación fue del 100% en las soluciones con 25.000 µg l‐1 de paracetamol; (ii) se
observaron anomalías sobre la movilidad de las larvas expuestas a concentraciones iguales o
superiores a 2.500 µg l‐1, pues estas permanecieron en posición lateral (23‐61%); (iii) en las
larvas expuestas a concentraciones iguales o superiores a 12.500 µg l‐1 se observaron espasmos o
movimientos involuntarios (26‐35%); y (iv) la longitud larvaria se incrementó ligeramente
(> 3%) en los embriones expuestos a concentraciones superiores a 6250 µg l‐1. Los resultados
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CAPÍTULO 9
indican que, tras la eclosión, el porcentaje y severidad de las anomalías se incrementó, haciéndose
más patente a las 144 hpf.
Figura 9.17 Tasas de mortalidad a 80 hpf de embriones de Danio rerio expuestos a soluciones experimentales de
paracetamol (a) y los efluentes procedentes de la biorremediación mediante microalgas (b). Nota: Las soluciones
experimentales de paracetamol se han nombrado como P_ seguido se la correspondiente concentración (µ l‐1). Los
efluentes procedentes de los sistemas de biorremediación tratando aguas contaminadas con paracetamol, se han
nombrado como PCS_, PCV_, y PSO_, para C. sorokiniana, C. vulgaris y S. obliquus, respectivamente, seguido del
porcentaje de dilución (50, 25). El control y el control del solvente fueron agrupados. Además, los tratamientos con la
misma concentración de diferentes experimentos (P_2500 y P_250) fueron agrupados. Las barras de error muestran
la desviación estándar (n= 12 para el control, P_2500 y P_250; n= 6 resto de grupos expuestos).
Los resultados obtenidos con los efluentes procedentes de la biorremediación de

paracetamol no mostraron una incidencia significativa sobre la mortalidad en ningún tiempo de
observación, al igual que ocurrió con las soluciones experimentales (Figura 9.17). Los mejores
resultados fueron los obtenidos mediante la biorremediación con C. sorokiniana, alcanzando una
eficiencia de eliminación de paracetamol del 67%. Esta eficiencia se reflejó en una reducción de
hasta el 62% de las anomalías totales a las 144 hpf, teniendo efectos tan solo sobre la
pigmentación (Tabla 3, Capítulo 8). Por otra parte, la peor eficiencia de eliminación (22%) fue en
el caso del tratamiento con C. vulgaris, el cual solo consiguió una reducción de las incidencia de las
anomalías totales de hasta el 31% a las 144 hpf. Las anomalías observadas se basaron en un
exceso de pigmentación y posición lateral (Tabla 3, Capítulo 8). En una posición intermedia se
encuentran los resultados obtenidos por la biorremediación mediante S. obliquus, que logró una
eliminación de paracetamol del 41%. En este caso, la incidencia de las anomalías se redujo hasta
un 56% a las 144 hpf respecto a la concentración inicial, mostrando estas larvas exceso de
pigmentación y posición lateral (Tabla 3, Capítulo 8).
168
9.5.2. Bioensayos con ácido salicílico
Los resultados obtenidos con las soluciones experimentales de ácido salicílico mostraron
una incidencia significativa sobre la mortalidad en los embriones expuestos a concentraciones
iguales o superiores a 12.500 µg l‐1 con tan solo 8 horas de exposición. La tasa de mortalidad
aumentó con el tiempo de exposición, llegando a mostrar diferencias significativas en la
concentración de 2.500 µg l‐1 a las 80 hpf, alcanzando entre el 20% y el 55% en función de la
concentración de ácido salicílico (Figura 9.18). En cuanto a las anomalías (Tabla 4, Capítulo 8),
a las 32 hpf los embriones expuestos a concentraciones iguales o superiores a 2.500 µg l‐1,
mostraron retraso significativo en el desarrollo y falta de pigmentación y un porcentaje
significativo de los coriones en contacto con 25.000 y 12.500 µg l‐1 se volvieron opacos y más
delicados a la manipulación. A las 80 hpf el porcentaje de anomalías no aumentó en gran medida
y tan solo se detectó un exceso de pigmentación, que se mantuvo a las 144 hpf. A la vista de los
resultados, se puede afirmar que el ácido salicílico causó una mayor incidencia en las primeras 32
horas de exposición.
ácido salicílico (a) y los efluentes procedentes de la biorremediación mediante microalgas (b). Nota: Las soluciones
experimentales de ácido salicílico se han nombrado como S_ seguido se la correspondiente concentración (µ l‐1). Los
efluentes procedentes de los sistemas de biorremediación tratando aguas contaminadas con ácido salicílico, se han
nombrado como SCS_, SCV_, y SSO_, para C. sorokiniana, C. vulgaris y S. obliquus, respectivamente, seguido del
porcentaje de dilución (50, 10). El control y el control del solvente fueron agrupados. Además, los tratamientos con la
misma concentración de diferentes experimentos (S_2500 y S_250) fueron agrupados. Las barras de error muestran
la desviación estándar (n= 12 para el control, S_2500 y S_250; n= 6 resto de grupos expuestos).
Los resultados obtenidos con los efluentes procedentes de la biorremediación de ácido

salicílico mostraron una incidencia significativa sobre la mortalidad a las 80 hpf para los
tratamientos al 50% de dilución con C. sorokiniana y C. vulgaris, tal y como se muestra en la
Figura 9.18. Los mejores resultados fueron los obtenidos con S. obliquus, alcanzando una
eficiencia de eliminación de ácido salicílico del 93%. Esta alta eficiencia quedó claramente
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CAPÍTULO 9
reflejada en los resultados de los ensayos embrionarios, ya que la mortalidad no mostró

diferencias significativas en ningún tiempo de observación y las anomalías observadas no fueron
relevantes (Tabla 4, Capítulo 8). Los resultados obtenidos por C. sorokiniana mostraron una
eficiencia intermedia entre las tres especies, alcanzando una eliminación de ácido salicílico del
73%. Aunque la tasa de mortalidad a las 80 hpf reflejó diferencias significativas (Figura 9.18),
ésta se redujo en un 55% respecto a la concentración inicial y las anomalías se redujeron en un
94% (Tabla 4, Capítulo 8). Finalmente, la biorremediación con C. vulgaris tan solo alcanzó una
eliminación de ácido salicílico del 25% y la tasa de mortalidad alcanzada no se redujo respecto a
la concentración inicial. Sin embargo, si lo hizo el porcentaje de anomalías, disminuyendo en un
33% las larvas que mostraron exceso de pigmentación (Tabla 4, Capítulo 8).
9.5.3. Bioensayos con diclofenaco
Los resultados obtenidos con las soluciones experimentales de diclofenaco mostraron que
la incidencia del fármaco sobre el desarrollo embrionario del pez cebra aumentó con la
concentración y a lo largo del tiempo de exposición. Como se puede ver en la Figura 9.19, los
efectos sobre la mortalidad fueron significativos a partir de las 80 hpf para concentraciones
iguales o superiores a 2.500 µg l‐1. Además, la mortalidad aumentó a las 144 hpf (Figura 6,
Capítulo 8), siendo total para las soluciones experimentales con concentraciones iguales o
superiores a 6.250 µg l‐1. Por otra parte, las anomalías (Tabla 2, Capítulo 8) comenzaron a
observarse a las 8 hpf, observándose un retraso del desarrollo en los embriones expuestos a
concentraciones iguales o superiores de 2.500 µg l‐1. Estas mismas concentraciones, a las 32 hpf
provocaron anomalías en casi la totalidad de los embriones, llegando a observarse anomalías en
cabeza, ojos, cola, saco vitelino, edema pericárdico y falta de pigmentación. Por lo tanto, el
diclofenaco fue capaz de causar graves anomalías antes de la eclosión. A las 80 hpf, la exposición a
concentraciones iguales o superiores a 6.250 µg l‐1 causó anomalías en el saco vitelino y edema
pericárdico en los embriones supervivientes. Finalmente, a las 144 hpf, los embriones
supervivientes (concentraciones de diclofenaco ≤ 2.500 µg l‐1) mostraron un exceso de
pigmentación y reducción de la movilidad, siendo esta última anomalía la detectada en los
embriones expuestos a concentraciones menores.
170
diclofenaco (a) y los efluentes procedentes de la biorremediación mediante microalgas (b). Nota: Las soluciones
experimentales de diclofenaco se han nombrado como D_ seguido se la correspondiente concentración (µ l‐1). Los
efluentes procedentes de los sistemas de biorremediación tratando aguas contaminadas con diclofenaco, se han
nombrado como DCS_, DCV_, y DSO_, para C. sorokiniana, C. vulgaris y S. obliquus, respectivamente, seguido del
porcentaje de dilución (50, 25 o 10). El control y el control del solvente fueron agrupados. Además, los tratamientos
con la misma concentración de diferentes experimentos (D_2500 y D_250) fueron agrupados. Las barras de error
muestran la desviación estándar (n= 12 para el control, D_2500 y D_250; n= 6 resto de grupos expuestos).
Los resultados obtenidos con los efluentes procedentes de la biorremediación de

diclofenaco mostraron una incidencia significativa sobre la mortalidad a las 80 hpf para los
tratamientos al 50% de dilución con C. sorokiniana y C. vulgaris, tal y como se muestra en la
Figura 9.19. En el caso de C. sorokiniana, tuvo una eficiencia de eliminación de diclofenaco del
66% y redujo las anomalías a las 32 hpf en un 26%, causando tan solo retraso en el desarrollo y
falta de pigmentación (Tabla 2, Capítulo 8). A pesar de que las anomalías a las 144 hpf fueron
significativas, la mortalidad se redujo en un 74% respecto a la concentración inicial de fármaco.
De manera similar, el tratamiento con C. vulgaris alcanzó una eliminación de diclofenaco del 69%.
Tanto la mortalidad como las anomalías a las 32 hpf (Tabla 2, Capítulo 8) fueron reducidas de
igual manera que en el caso de C. sorokiniana y la mortalidad a las 144 hpf se redujo en un 67%.
Sin embargo, la biorremediación con S. obliquus alcanzó los mejores resultados, logrando una
eficiencia de eliminación del 99%. La mortalidad no resultó significativa a ningún momento de
observación, mostrando tan solo un 30,7% de las larvas con movilidad reducida a las 144 hpf
(Tabla 2, Capítulo 8).
Existen numerosos estudios, basados en diferentes tipos de ensayos, sobre la toxicidad de

fármacos. De los fármacos considerados en este trabajo, los efectos sobre los embriones del pez
cebra (Danio rerio) fueron estudiados por Ribeiro et al. (2015) para el diclofenaco (12,5 a
12.500 µg l‐1). Además, los efectos del paracetamol sobre el desarrollo embrionario del pez cebra
fueron estudiados por David y Pancharatna (2009) para concentraciones entre 1 y 100 µg l‐1. Sin
embargo, no tenemos conocimiento de que existan estudios anteriores sobre los efectos
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CAPÍTULO 9
toxicológicos de los productos de transformación del diclofenaco, paracetamol y ácido salicílico

tras el proceso de biorremediación de microalgas sobre el desarrollo embrionario de Danio rerio.
Además, hasta donde sabemos, no existe información sobre la capacidad de las microalgas para
reducir la toxicidad de aguas contaminadas con fármacos. En esta tesis, los sistemas de
biorremediación mediante C. sorokiniana, C. vulgaris y S. obliquus no solo fueron capaces de
reducir la concentración de fármacos en el agua tratada, sino también su toxicidad. El estudio de
los efectos sobre el desarrollo embrionario de Danio rerio mostró que la toxicidad de los efluentes
fue equivalente o menor que la determinada para la misma concentración en la soluciones
experimentales. Por lo tanto, la eliminación de paracetamol, ácido salicílico y diclofenaco
mediante estas especies de microalgas no dio lugar a productos de transformación tóxicos, lo que
es de suma importancia de cara a su aplicación en la biorremediación de aguas contaminadas con
fármacos.
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176
Capítulo 10
Conclusiones
Conclusiones
10.1. CONCLUSIONES
A partir del trabajo realizado, cuyos resultados se encuentran recogidos en detalle en las
publicaciones que conforman los Capítulos 4‐8, las principales conclusiones que se pueden
extraer son:
i. La presencia de los fármacos paracetamol, ácido salicílico y diclofenaco no tuvo

efectos negativos sobre el crecimiento de las especies C. sorokiniana, C. vulgaris y
S. obliquus. Excepto en el caso de S. obliquus, que no hubo diferencias significativas, la
presencia del fármaco supuso un incremento en la concentración de biomasa,
pudiéndose explicar por el hecho de que los fármacos constituyeron una fuente
adicional de carbono.
ii. La eliminación de nutrientes mediante microalgas al final del cultivo discontinuo fue
casi completa tanto en presencia como en ausencia de fármacos. Sin embargo,
durante el estado estacionario del cultivo semicontinuo, S. obliquus fue la especie
que alcanzó las mayores eficiencias de eliminación de nutrientes. Las tres especies
de microalgas estudiadas mostraron una mayor eficiencia de eliminación de fosfatos
que de nitratos, lo que se asocia a una limitación de fósforo en el cultivo
semicontinuo. De cualquier forma, fue probado que la presencia de fármacos supuso
una mayor eficiencia en la eliminación de nutrientes como consecuencia del mayor
crecimiento de las microalgas asociado.
iii. Los resultados obtenidos en el eliminación de paracetamol al final del cultivo

discontinuo evidenciaron una mayor capacidad mediante C. sorokiniana, mostrando
eficiencias del 67% de una concentración inicial de 25 mg l‐1. Sin embargo, partiendo
de esa misma concentración, S. obliquus fue la especie que alcanzó la mayor
eliminación en el caso del ácido salicílico y diclofenaco, logrando eficiencias
superiores al 93% y 98%. En el estado estacionario del cultivo semicontinuo las
eficiencias de eliminación fueron menores que en el cultivo discontinuo. Sin
embargo, al igual que en el cultivo discontinuo, S. obliquus continuó siendo la especie
que alcanzó los mejores resultados en la eliminación de ácido salicílico y
diclofenaco, mientras que C. sorokiniana lo fue en el caso del paracetamol.
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CAPÍTULO 10
iv. El cultivo de C. sorokiniana en altas concentraciones relativas (250 mg l‐1) de

paracetamol y ácido salicílico no solo demostró la resistencia de la especie sino
también la capacidad de alcanzar mayores eliminaciones netas, logrando al final del
cultivo discontinuo eficiencias superiores al 94% en la eliminación de ácido salicílico
y un 48% en el caso del paracetamol. De la misma manera, en el estado estacionario
del cultivo semicontinuo, la eliminación por gramo de biomasa de estos fármacos
fue de un orden de magnitud superior que la alcanzada para concentraciones de
25 mg l‐1.
v. Después del tratamiento, los polielectrolitos sintéticos resultaron ser

comparativamente más rápidos y eficientes en la separación de la biomasa de
microalgas, aunque la viscosidad de los flóculos obtenidos puede suponer una
limitación en el posterior uso de misma. Las sales metálicas resultaron ser una
alternativa eficiente, obteniéndose mayores eficiencias de separación mediante la
sal de aluminio para C. sorokiniana y C. vulgaris. Es de destacar que, en todos los
casos, las eficiencias de separación con la biomasa procedente del cultivo
semicontinuo fueron inferiores que con la biomasa del discontinuo; lo que evidencia
la influencia de la edad del cultivo en la eficiencia del floculante.
vi. Entre los tres fármacos considerados, los efectos más severos sobre el desarrollo
embrionario de Danio rerio, en cuanto a mortalidad y anomalías morfológicas,
fueron los producidos por el fármaco diclofenaco. En cualquier caso, la
biorremediación mediante microalgas resultó en una disminución de los efectos
negativos producidos por los tres fármacos. En este sentido, tras el tratamiento, la
especie S. obliquus fue capaz de reducir la mortalidad y las anomalías producidas por
la presencia de ácido salicílico, igualándola a los valores obtenidos en ausencia del
fármaco. Igualmente, S. obliquus fue capaz de reducir la mortalidad producida por el
diclofenaco desde valores del 100% hasta el 12%, presentando anomalías tan solo
en el 31% de los casos. En el caso del paracetamol, fue la especie C. sorokiniana la
que resultó ser más eficiente, reduciendo las anomalías en un 62%. Se puede afirmar
por tanto, que los sistemas de biorremediación mediante C. sorokiniana, C. vulgaris y
S. obliquus, además de disminuir la concentración de los fármacos, fueron capaces de
reducir la toxicidad de los efluentes tratados.
180
Conclusiones
10.2. CONCLUSIONS
On the basis of the study carried out, whose results are presented in detail in the
publications comprised within Chapters 4‐8, the main conclusions of this work are:
i. The presence of the pharmaceuticals paracetamol, salicylic acid and diclofenac did
not have negative effects on the growth of the strains C. sorokiniana, C. vulgaris and
S. obliquus. Except in the case of S. obliquus, which did not showed significant
differences, the presence of pharmaceuticals increased the biomass concentration,
which may be explained by the fact that pharmaceuticals were an additional source
of carbon.
ii. At the end of the batch culture, the removal of nutrients by microalgae was nearly
complete both in the presence and in the absence of pharmaceuticals. However, at
the steady state of the semicontinuous culture, S. obliquus was the strain that
reached the highest efficiencies in the removal of nutrients. The three studied
microalgae strains showed higher removal of phosphates than nitrates, which is
associated with a phosphorous limitation at the semicontinuous culture. In any case,
it was proved that the presence of pharmaceuticals increased the removal of
nutrients as a result of the higher microalgae growth associated.
iii. The results obtained in the removal of paracetamol at the end of the batch culture,
highlighted the higher removal ability by C. sorokiniana, which showed efficiencies
above 67% from a 25 mg l‐1 initial concentration. However, from this concentration,
S. obliquus was the strain that obtained the highest efficiency in the case of salicylic
acid and diclofenac, reaching efficiencies higher than 93% and 98%, respectively.
At the steady state of the semicontinuous culture, the removal efficiencies were
lower than at the batch culture. However, as at the batch culture, S. obliquus was the
strain that achieved the best results in the removal of salicylic acid and diclofenac,
meanwhile C. sorokiniana in the case of paracetamol.
181
CAPÍTULO 10
iv. C. sorokiniana culture in relative high concentrations (250 mg l‐1) of paracetamol

and salicylic acid proved not just the robustness of the strain but also the ability to
attain higher net removals, reaching efficiencies higher than 94% for salicylic acid
and 48% for paracetamol at the end of the batch culture. Likewise, at the steady
state of the semicontinuous culture, the removal of these drugs per gram of biomass
was one order of magnitude higher than those reached from 25 mg l‐1
concentrations.
v. After treatment, the synthetic polyelectrolytes were comparatively quicker and

more efficient in the recovery of microalgae biomass, although the viscosity of the
flocs obtained may involve a limitation in the subsequent use of the biomass. Metal
salts proved to be an efficient alternative, with higher recovery efficiencies by
aluminum salt in C. sorokiniana and C. vulgaris biomass. It should be noted that, in all
cases, the recovery efficiencies of the biomass from the semicontinuous culture were
lower than those obtained from the batch culture; which evidence the influence of
the age of the culture in the flocculant efficiency.
vi. Among the three considered pharmaceuticals, the most severe effects on the
embryonic development of Danio rerio, namely on mortality and morphological
abnormalities, were caused by diclofenac. In any case, bioremediation by microalgae
resulted in a reduction of the negative effects caused by the three pharmaceuticals.
In this sense, after treatment, the strain S. obliquus was able to reduce the mortality
and abnormalities produced by salicylic acid, so they were equal to the values
obtained in absence of this drug. Likewise, S. obliquus was able to reduce the
mortality produced by diclofenac from 100% to 12%, in which only 31% of the cases
showed abnormalities. In the case of paracetamol, C. sorokiniana was the most
efficient, reducing the abnormalities up to 62%. It is therefore affirmed that,
bioremediation systems by C. sorokiniana, C. vulgaris and S. obliquus, besides the
reduction of the pharmaceuticals concentration, allowed for the decrease of the
toxicity in the effluents.
182

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BIORREMEDIACIÓN DE AGUAS CONTAMINADAS CON NUTRIENTES Y FÁRMACOS MEDIANTE MICROALGAS

BIORREMEDIACIÓN DE AGUAS CONTAMINADAS

Carla Escapa Santos

BIORREMEDIACIÓN DE AGUAS CONTAMINADAS CON

Bioremediation of contaminated waters

Nutrients and pharmaceuticals removal from wastewater by culture and harvesting of

Escapa C, Coimbra R.N, Paniagua S, García A.I, Otero M

Bioresource Technology, 185 (2015): 276‐284

Paracetamol and salicylic acid removal from contaminated water by microalgae

Escapa C, Coimbra R.N, Paniagua S, García A.I, Otero M

Journal of Environmental Management, (2016) doi: 10.1016/j.jenvman.2016.06.051

Escapa C, Coimbra R.N, Paniagua S, García A.I, Otero M

Journal of Applied Phycology, (2016) doi: 10.1007/s10811‐016‐1010‐5

Comparative assessment of diclofenac removal from water by different microalgae strains

Escapa C, Coimbra R.N, Paniagua S, García A.I, Otero M

Algal Research, 18 (2016): 127‐134

Ability of microalgae bioremediation systems to reduce the toxicity of pharmaceuticals

Escapa C, Torres T, Neuparth T, Santos M.M, García A.I, Otero M

Enviado a Water Reseach (marzo de 2017)

A todos ellos, gracias.

La eficiencia en la eliminación de nitratos y fosfatos de las especies de microalgas antes

Para abordar la eficiencia de eliminación de los fármacos paracetamol, ácido salicílico y

Finalmente, con el fin de evaluar la capacidad de las microalgas en la disminución de la

1.1 JUSTIFICACIÓN DE LA UNIDAD TEMÁTICA

La preocupación por la presencia de productos farmacéuticos en el medio acuático ha llevado

Agua (2000/60/CE) . Fue en la Propuesta de la Comisión Europea de 31 de enero de 2012 , cuando se

estradiol (E2) y 17‐alfa‐etinilestradiol (EE2). Finalmente, la Decisión de la UE 2015/495 , incluyó

1.2. PRESENTACIÓN DE LAS PUBLICACIONES

La presente tesis es un compendio de cinco publicaciones en las que se estudian diferentes

En el Capítulo 4 (Artículo I) “Nutrients and pharmaceuticals removal from wastewater by

En el Capítulo 6 (Artículo III) “Comparison of the culture and harvesting of Chlorella

En el Capítulo 8 (Artículo IV) “Comparative assessment of diclofenac removal from water

En el Capítulo 9 (Artículo V) “Ability of microalgae bioremediation systems to reduce the

2.1. LA PROBLEMÁTICA DE LAS AGUAS RESIDUALES

2.2. LOS CONTAMINANTES EMERGENTES

El reciente desarrollo de nuevos y más sensible métodos analíticos, ha permitido identificar

La presencia de estos contaminantes en aguas naturales está relacionada principalmente

A pesar de haberse registrado más de 10.000 sustancias potencialmente peligrosas en la

Tabla 2.1 Principales contaminantes emergentes (García-Navas, 2013).

Antibióticos, analgésicos, antiinflamatorios, antidepresivos,

Drogas de abuso Cocaínicos, anfetamínicos, cannabinoides, opiáceos, alucinógenos

Hormonas esteroideas Estradiol, estrona, estriol, dietilestilbestrol

Fragancias, nitropolicíclicos, macrocíclicos, parabenos,

Agentes para la protección solar Benzofenona, metilbenzilideno

Antisépticos Triclosán o clorofeno

Agentes de limpieza y detergentes Alquifenoles y carboxilados alquifenoles

Retardantes de llama Éteres polibromados

Agentes y aditivos industriales 2-cloroetilfosfato

Aditivos para la gasolina Éteres de aquilos o éter metil-t-butilo

Subproductos de la desinfección Bromoácidos, bromoacetonitrilos, bromatos

Otras sustancias Nicotina, cafeína

En las últimas décadas, muchas organizaciones de reconocida relevancia han tomado

El siguiente paso, comprende la evaluación del destino y biodisponibilidad de estos

Por último, otra de las líneas de investigación prioritarias se centra en el estudio de

2.3. LOS FÁRMACOS COMO CONTAMINANTES EMERGENTES

Las primeras evidencias de la presencia de fármacos en el agua se remontan a los años 70

La entrada de contaminantes de tipo farmacológico al medio acuático puede deberse a los

Por otra parte, las aguas residuales procedentes de la industria farmacéutica se

En función de las propiedades físico-químicas de los fármacos, metabolitos y/o productos

Recientes estudios han demostrado que los sistemas de tratamiento convencionales

Además de que los tratamientos de aguas residuales convencionales no hayan sido

• Antiinflamatorios y analgésicos. Dentro de este grupo, los compuestos más

2.4. EFECTOS Y RIESGOS DE LA PRESENCIA DE FÁRMACOS EN EL MEDIO AMBIENTE