Biorremediación de suelos - URJC

Biorremediacioacuten de suelos contaminados con hidrocarburos

aromaacuteticos policiacuteclicos

Raquel Simarro Doblado

Dra Natalia Gonzaacutelez y Dra Mariacutea del Carmen Molina profesoras titulares del

Departamento de Biologiacutea y Geologiacutea de la Universidad Rey Juan Carlos

CERTIFICAN

Que los trabajos de investigacioacuten desarrollados en la memoria de tesis doctoral

ldquoBiorremediacioacuten de suelos contaminados con hidrocarburos aromaacuteticos policiacuteclicosrdquo son aptos para ser presentados por la Lda Raquel Simarro Doblado ante el

Tribunal que en su diacutea se consigne para aspirar al Grado de Doctor en Ciencias

Ambientales por la Universidad Rey Juan Carlos de Madrid

VordmBordm Director Tesis VordmBordm Director de Tesis

Dra Natalia Gonzaacutelez Beniacutetez Dra Mordf Carmen Molina

A mi familia a Javi y amigos todos ellos forman parte de esta tesis como si de un capiacutetulo se tratase

A todos gracias por formar parte de los capiacutetulos de mi vida

Iacutendice

I Resumen Antecedentes 13 Objetivos 25 Listado de manuscritos 27 Siacutentesis de capiacutetulos 29 Metodologiacutea general 33

Capiacutetulo 1a Optimisation of key abiotic factors of PAH (naphthalene phenanthrene

and anthracene) biodegradation process by a bacterial consortium 47

b Evaluation of the influence of multiple environmental factors on the biodegradation of dibenzofuran phenanthrene and pyrene by a bacterial consortium using an orthogonal experimental design 67

Capiacutetulo 2 Effect of surfactants on PAH biodegradation by a bacterial consortium

and on the dynamics of the bacterial community during the process 85

Capiacutetulo 3 High molecular weight PAH biodegradation by a wood degrading

consortium at low temperatures 113

Capiacutetulo 4 Assessment the efficient of bioremediation techniques (biostimulation

bioaugmentation and natural attenuation) in a creosote polluted soil change in bacterial community 143

II Discusioacuten general 171

III Conclusiones generales 181

IV Referencias bibliograacuteficas 185

V Agradecimientos 195

Resumen

AntecedentesObjetivos

Listado de manuscritosSiacutentesis de capiacutetulosMetodologiacutea general

I

Resumen Antecedentes

13

Antecedentes

Tal y como su propio nombre indica biorremediacioacuten significa ldquodar remediordquo mediante

teacutecnicas bioloacutegicas por lo que una definicioacuten acertada de biorremediacioacuten seriacutea el conjunto

de teacutecnicas que permiten resolver problemas de contaminacioacuten mediante el uso de

microorganismos (bacterias algas unicelulares etc) hongos plantas o enzimas derivadas

de ellos En el contexto de esta tesis la biorremediacioacuten se aplica a suelos u otros sistemas

contaminados con hidrocarburos aromaacuteticos policiacuteclicos (HAP o PAH siglas en ingleacutes

polyciclic aromatic hydrocarbons) Los HAP son compuestos que se generan por la

combustioacuten incompleta de combustibles foacutesiles por causas naturales y en mayor medida

antroacutepicas Si tenemos en cuenta que el sustento del sistema energeacutetico mundial son los

combustibles foacutesiles podemos hacernos una idea de la relevancia y elevada presencia de

estos compuestos en la vida cotidiana Considerando sus complejas propiedades y su

caraacutecter perjudicial para el ser humano y los ecosistemas (Internacional Agency for

Research on Cancer 1972-1990) los HAP suscitaron una creciente preocupacioacuten a partir

del pasado siglo siendo los primeros carcinoacutegenos ambientales reconocidos (Haritash amp

Kaushik 2009) Eacutesto dio lugar a numerosos estudios cientiacuteficos con el objetivo de

determinar sus efectos en el medio y el ser humano asiacute como a la buacutesqueda de soluciones

para su eliminacioacuten Frente a muchas otras teacutecnicas de eliminacioacuten de contaminantes

(teacutecnicas fiacutesicas yo quiacutemicas) la biorremediacioacuten presenta una caracteriacutestica clave que la

hace destacar sobre las otras teacutecnicas y es que se basa en procesos que respetan el medio

perturbado y permiten en la medida de lo posible su recuperacioacuten

Los hidrocarburos aromaacuteticos policiacuteclicos (HAP) Legislacioacuten espantildeola sobre medios

contaminados

La presente tesis se centra en el estudio de la biodegradacioacuten de los hidrocarburos

aromaacuteticos policiacuteclicos y por ello es esencial conocer a fondo queacute son cuaacuteles son sus

caracteriacutesticas y porqueacute suscitan este intereacutes Los HAP son compuestos quiacutemicos formados

por la fusioacuten de un determinado nuacutemero de anillos de su principal componente aromaacutetico el

benceno La estructura quiacutemica de alguno de los compuestos que han sido utilizados

durante el desarrollo de esta tesis aparecen en la Figura 1


14

Figura 1 Hidrocarburos aromaacuteticos poliacuteciacuteclicos de bajo (naftaleno fenantreno y antraceno) y alto peso

molecular (pireno y perileno)

Los HAP se pueden clasificar en funcioacuten del nuacutemero de anillos benceacutenicos en HAP de

bajo (menos de tres anillos) y de alto peso molecular Tan soacutelo el naftaleno fenantreno y

antraceno (dos y tres anillos respectivamente) seriacutean considerados HAP de bajo peso

molecular (Cerniglia 1984 1992) La propiedades quiacutemicas de los HAP y por tanto su

destino en el medioambiente depende del nuacutemero de anillos aromaacuteticos que lo componen y

de su tipologiacutea molecular (Kanaly amp Harayama 2000) Por ejemplo el fenantreno y

antraceno son compuestos isoacutemeros (Figura 1) con el mismo peso molecular porque tienen

el mismo nuacutemero de anillos benceacutenicos pero en distinta disposicioacuten lo que les confiere

distintas caracteriacutesticas quiacutemicas En general a medida que aumenta su tamantildeo peso

molecular y angularidad aumenta su hidrofobicidad y estabilidad electroquiacutemica (Zander

1983) Tal y como algunos trabajos han demostrado eacutestos son dos factores primarios que

contribuyen a la persistencia de los HAP en el medio Por ejemplo de acuerdo con

Shuttleworth amp Cerniglia (1995) la vida media en suelos y sedimentos de un HAP de tres

anillos como el fenantreno podriacutea oscilar en un rango de entre 6 a 126 diacuteas mientras que

para moleacuteculas de cuatro a cinco anillos el tiempo aumentariacutea de 229 a maacutes de 1400 diacuteas

Ademaacutes debido a su naturaleza lipofiacutelica tiene un elevado potencial de bioacumulacioacuten en la

cadena troacutefica fenoacutemeno conocido como biomagnificacioacuten (Clements et al 1994) Se sabe

que los HAP ejercen un acusado efecto toacutexico y poseen propiedades mutageacutenicas

teratogeacutenicas y en algunos casos carcinogeacutenicas (Internacional Agency for Research on

Cancer 1972-1990) De hecho dieciseacuteis HAP han sido clasificados como contaminantes

prioritarios por la US Environmental Protection Agency (Agencia de proteccioacuten ambiental


15

de Estados Unidos) y por la Comisioacuten Europea de Medio Ambiente (Maliszewska-Kordybach

1996)

Los HAP estaacuten presentes como constituyentes naturales de los combustibles foacutesiles y

se forman durante la combustioacuten incompleta de la materia orgaacutenica Las fuentes naturales

de produccioacuten de HAP son los incendios forestales y de pastizales yacimientos de petroacuteleo

o erupciones volcaacutenicas (Haritash amp Kaushik 2009) Sin embargo las fuentes antroacutepicas

son las que maacutes contribuyen a su formacioacuten mediante la quema de combustibles foacutesiles con

fines energeacuteticos en el tratamiento de la madera con creosota mediante el uso de

lubricantes y en el refino del petroacuteleo y actividades de transporte (Lee et al 1981) Aunque

los vertidos se produzcan en una zona determinada es posible que la carga contaminante

se extienda si alcanza un efluente o mediante su filtracioacuten a traveacutes del suelo pudiendo

alcanzar acuiacuteferos Otras cargas contaminantes se generan por descarga directa

procedentes de efluentes industriales en grandes superficies de suelos o mares o por la

liberacioacuten accidental de materia prima (Kanaly amp Harayama 2000) Se han detectado HAP

en el aire procedente fundamentalmente de la quema industrial de combustibles foacutesiles y el

traacutefico (Koeber et al 1999 Lim et al 1999) en suelos y en la parte superficial y profunda

de la columna de agua y sedimentos (Readman et al 2002 Johnsen et al 2005 2006) En

alimentos vegetales y plantas aparecen como consecuencia de la difusioacuten y posterior

sedimentacioacuten de los HAP atmosfeacutericos sobre la vegetacioacuten (Wagrowski amp Hites 1997) y

por la adsorcioacuten de HAP acumulados en el agua del suelo

El suelo constituye uno de los medios receptores de la contaminacioacuten maacutes sensibles y

vulnerables Sin embargo y auacuten dada la gravedad y extensioacuten creciente de la contaminacioacuten

con HAP no fue hasta la Conferencia de Naciones Unidas sobre el Medio Ambiente y el

Desarrollo celebrada en Riacuteo de Janeiro en 1992 cuando se reconocioacute y planteoacute de forma

trascendente la importancia de la proteccioacuten de los suelos y la definicioacuten de sus usos

potenciales La Agencia Europea de Medio Ambiente (AEMA) estimoacute en 1999 que el

nuacutemero de zonas o aacutereas contaminadas en Europa Occidental estaba entre 300000 y

1500000

Hasta la promulgacioacuten de la Ley 101998 de 21 Abril de Residuos Espantildea careciacutea de

cualquier instrumento normativo para legislar controlar y proteger lo referente a suelos

contaminados Con esta Ley las Comunidades Autoacutenomas son las encargadas de declarar

delimitar e inventariar los suelos contaminados de sus territorios ademaacutes de establecer las

bases para una determinada actuacioacuten Con el Real decreto 92005 del 14 de Enero se da

cumplimiento a lo dispuesto en la 101998 de Residuos y en eacutel se establece la relacioacuten de

actividades potencialmente contaminantes del suelo y los criterios y estaacutendares para la


16

declaracioacuten de un suelo bajo la categoriacutea de ldquocontaminadordquo Uno de los aspectos maacutes

importantes desde el punto de vista de la biorremediacioacuten se recoge en el Artiacuteculo 7 del

Real Decreto 92005 por el cual ldquola declaracioacuten de un suelo como contaminado obligaraacute a la

realizacioacuten de las actuaciones necesarias para proceder a su recuperacioacuten ambientalrdquo

Ademaacutes antildeade que las teacutecnicas deben ser las maacutes apropiadas en cada caso garantizando

soluciones de caraacutecter permanente y prioriza las teacutecnicas de tratamiento in situ que eviten la

generacioacuten traslado y eliminacioacuten de residuos

Lo dispuesto en esta ley sin duda supone un enorme impulso a las teacutecnicas de

biorremediacioacuten ya que a traveacutes de ellas se favorece el tratamiento in situ y la recuperacioacuten

del ecosistema a su estadio original sin generacioacuten de residuos y con el menor impacto

ambiental posible

Factores que condicionan la biodegradacioacuten

Aunque la biodegradacioacuten bacteriana es una de las teacutecnicas maacutes eficaces en la

descontaminacioacuten in situ de medios contaminados con HAP la eficacia y tasas de

biodegradacioacuten dependen en gran medida del nuacutemero y tipo de microorganismo

degradador presente en el medio y de la naturaleza y estructura quiacutemica del contaminante a

degradar (Haritash amp Kaushik 2009) Ademaacutes a la hora de disentildear un sistema de

biorremediacioacuten debemos tener en cuenta que existen muacuteltiples factores ambientales que

van a condicionar la eficacia y la rapidez del proceso con el agravante de que durante la

aplicacioacuten in situ es difiacutecil y en algunos casos imposible poder controlar o modificar alguno

de estos factores La biorremediacioacuten es una teacutecnica eficaz y econoacutemica pero cuenta con la

desventaja del tiempo ya que en algunos casos la descontaminacioacuten del medio y su

recuperacioacuten pueden durar antildeos

Por tanto los estudios de optimizacioacuten en los que se combinen todos los factores

posibles considerando los efectos sineacutergicos y antagoacutenicos son esenciales en

biorremediacioacuten Entre los factores ambientales maacutes estudiados en la literatura destacamos

temperatura pH tipo y concentracioacuten de nutrientes inorgaacutenicos y fuentes de carbono

Temperatura y pH

La temperatura es una de las variables maacutes influyentes en el proceso de biodegradacioacuten

bacteriana ya que afecta tanto a las propiedades fisicoquiacutemicas de los HAP como al


17

metabolismo microbiano La temperatura guarda una relacioacuten proporcional con los rangos

de difusioacuten y solubilidad de los HAP e inversamente proporcional con el coeficiente de

particioacuten suelo-agua (Wu amp Gschwend 1986) Mientras que el coeficiente de particioacuten de los

HAP decrece entre un 20-30 por cada incremento de 10ordmC en temperaturas comprendidas

entre los 5 ordmC - 45 ordmC el coeficiente de difusioacuten en agua aumenta entre 4-5 veces con un

incremento de la temperatura de 20 a 120 ordmC Esto se traduce en que cuanto menor es la

temperatura menor es la solubilidad de los HAP en la fase acuosa y consecuentemente

menor es su biodisponibilidad para ser mineralizados por los microorganismos (Haritash amp

Kaushik 2009)

Por otro lado las bajas temperaturas afectan negativamente al metabolismo

microbiano ralentizaacutendolo y aumentando la duracioacuten de la fase de latencia en la que hay

inactividad (Atlas amp Bartha 1972 Eriksson et al 2001) Tal y como se ha demostrado en

estudios previos (Leahy amp Colwell 1990) la velocidad de metabolizacioacuten normalmente se

duplica por cada aumento de 10 ordmC en temperaturas comprendidas entre los 10 y 40 ordmC Sin

embargo y a pesar de las desventajas que las bajas temperaturas presentan para la

biodegradacioacuten existe degradacioacuten de hidrocarburos en ambientes friacuteos cuyas temperaturas

oscilan entre los 0 ordmC ndash 10 ordmC (Margesin et al 2002) Algunos trabajos se han centrado en el

estudio de la biodegradacioacuten en zonas de agua marina y suelos bajo temperaturas

extremadamente bajas (Colwell et al 1978 Mohn amp Stewart 2000 Ericksson et al 2001

Delille amp Pelletiere 2002) obteniendo resultados positivos Sin embargo la mayoriacutea de los

estudios de biodegradacioacuten se han llevado a cabo en condiciones de laboratorio en un rango

de temperaturas comprendido entre los 20 ndash 35 ordmC sin observar a penas diferencias en las

tasas de biodegradacioacuten (Chen et al 2008) Por debajo de estas temperaturas la

degradacioacuten es maacutes complicada porque el metabolismo de los microorganismos se ralentiza

y la solubilizacioacuten de los HAP disminuye Aun asiacute hay bacterias adaptadas a estas

condiciones que hacen posible la degradacioacuten en ambientes con temperaturas extremas

Varias especies de Pseudomonas y Sphingomonas se han identificado como bacterias

degradadoras de HAP en la Antaacutertida (Aislabie et al 2000) Eacutestas y otras especies estaacuten

adaptadas a las temperaturas locales y a otras condiciones de estreacutes ya que durante el

deshielo sobreviven en suelos friacuteos y secos pobres en nutrientes y a menudo alcalinos Sin

embargo la capacidad de estas bacterias para crecer en suelos con condiciones climaacuteticas

suaves y la utilizacioacuten de HAP para su crecimiento implica que estas bacterias son

psicrotolerantes (Aislabie et al 2000) cuya temperatura oacuteptima es superior a los 20 ordmC pero

son capaces de sobrevivir cerca o por debajo de los 0 ordmC Ademaacutes existen algunas especies

cuyo crecimiento se desarrolla en ambientes que permanentemente esteacuten por debajo de los

5 ordmC este grupo de bacterias son psicroacutefilas La temperatura es un factor que difiacutecilmente se

puede controlar o manipular en proyectos de aplicacioacuten in situ por lo que es importante


18

elaborar un estudio previo bajo las condiciones del ecosistema afectado Esto es

fundamental sobre todo en ambientes con temperaturas bajas en los que se puede estudiar

queacute otros factores modificar para suplir las desventajas de la temperatura como puede ser

inocular cepas bacterianas adaptadas a las bajas temperaturas aumentar el oxiacutegeno o

adicionar nutrientes En estas condiciones no toda la comunidad tiene por queacute ser eficaz en

la degradacioacuten de un contaminante aunque se ha propuesto que las bacterias y no los

hongos son las mayores colonizadoras y degradadoras (Kerry 1990) La identificacioacuten de

las especies de la poblacioacuten autoacutectona asiacute como la refrenciacioacuten de sus capacidades

metaboacutelicas son muy importantes en la biorremediacioacuten de suelos de ambientes friacuteos Esta

cuestioacuten es especialmente importante en la Antaacutertida ya que el Sistema del Tratado

Antaacutertico prohiacutebe la introduccioacuten de organismos aloacutectonos

Por otro lado el pH es un factor abioacutetico que de forma similar a la temperatura puede

afectar significativamente tanto a la actividad y diversidad microbiana como a la

mineralizacioacuten de los HAP Los rangos de pH oacuteptimos para el proceso de metabolizacioacuten

pueden ser muy variables ya que depende de las caracteriacutesticas del medio contaminado y

de la poblacioacuten microbiana que alberga (Dibble amp Bartha 1979) Las micobacterias son

bacterias neutroacutefilas cuyo pH oacuteptimo es superior 6 (Portaels amp Pattyn 1982) Sin embargo

a paritr de este pH se ha observado que la degradacioacuten de HAP por Mycobacterium es maacutes

eficaz cuando el pH tiende ligeramente a aacutecido (65) porque la membrana de aacutecidos

micoacutelicos es maacutes permeable a compuestos hidrofoacutebicos (Kim et al 2005) Otros autores

han mostrado que para otro tipo de bacterias comuacutenmente descritas en procesos de

biodegradacioacuten como Pseudomonas sp el rango oacuteptimo de pH oscila entre 55 y 78

notablemente mejor cuanto maacutes neutro (Dibble amp Bartha 1979) El uso de ciertos

surfactantes puede causar la basificacioacuten del medio (Bautista et al 2009) por lo que este

aspecto debe ser considerado a la hora de disentildear un proceso de biorremediacioacuten Tambieacuten

se pueden generar variaciones de pH durante el proceso como consecuencia de los

metabolitos intermedios derivados de los HAP Por ejemplo al comienzo de la degradacioacuten

se generan metabolitos con grupos hydroxiacutelicos que producen un aumento del pH (Habe amp

Omori 2003 Puntus et al 2008)

Nutrientes inorgaacutenicos

Ante una perturbacioacuten por un contaminante el requerimiento de nutrientes de las bacterias

degradadoras del ecosistema aumenta para poder metabolizar el aporte extra de carbono

que supone el contaminante Por este motivo en biorremediacioacuten es importante encontrar

una relacioacuten oacuteptima de carbononitroacutegenofoacutesforo (CNP) que tradicionalmente se han fijado


19

en 100101 (ej Bouchez et al 1995) Sin embargo este aspecto es objeto de controversia

ya que otros autores (Leys et al 2005) han demostrado que la relacioacuten anteriormente

propuesta como oacuteptima puede ser insuficiente limitando el crecimiento bacteriano y por

tanto ralentizando la biodegradacioacuten La bioestimulacioacuten es una teacutecnica de biorremediacioacuten

que consiste en la adicioacuten de nutrientes inorgaacutenicos a los substratos contaminados La

disponibilidad de nutrientes es un aspecto muy importante en la eficacia de la

biodegradacioacuten Nutrientes como el nitroacutegeno el foacutesforo o el hierro son esenciales para el

metabolismo bacteriano en general y mucho maacutes en el caso de biorremediacioacuten de medios

contaminados por HAP Aunque la mayoriacutea de los trabajos indican que la adicioacuten de

nutrientes mejora el proceso algunos autores (Yu et al 2005) obtuvieron resultados

opuestos La diferencia entre unos resultados y otros radican en que la necesidad de

nutrientes depende del tipo de bacteria tipo de hidrocarburo y de las condiciones del medio

(Leys et al 2005) El hierro es un nutriente esencial necesario en el proceso de

biodegradacioacuten ya que por un lado es cofactor de las enzimas que catalizan la oxidacioacuten de

los HAP y por otro se ha relacionado con la produccioacuten de biosurfactantes para potenciar la

solubilidad de los HAP (Wei et al 2003) Sin embargo determinar la proporcioacuten oacuteptima de

este nutriente es fundamental pues altas concentraciones pueden ser toacutexicas (Santos et al

2008) Es tambieacuten una cuestioacuten a considerar la forma en la que se adicionan o se

encuentran los nutrientes en el medio ya que condiciona su biodisponibilidad Asiacute algunos

autores (Schlessinger 1991) proponen que las formas oxidadas como nitratos son maacutes

solubles que las formas reducidas como amonio que ademaacutes tiene propiedades

adsorbentes Establecer si un determinado problema medioambiental requiere un aporte

exoacutegeno de nutrientes es por tanto una cuestioacuten a discutir que probablemente dependa de

otras variables bioacuteticas y abioacuteticas

Fuentes de carbono laacutebiles

La adicioacuten a un medio contaminado de otras fuentes de carbono faacutecilmente biodegradables

se considera una alternativa que puede favorecer la biodegradacioacuten porque aumenta la

biomasa de la poblacioacuten microbiana y por tanto de la poblacioacuten degradadora Realmente se

puede entender como una forma de bioestimulacioacuten enfocada a aumentar y estimular el

crecimiento bacteriano o su actividad y por consiguiente la degradacioacuten Algunas de las

sustancias tratadas con este fin son el piruvato que estimula el crecimiento de ciertas cepas

bacterianas o el salicilato que induce la activacioacuten de enzimas degradadoras En el caso de

la glucosa su raacutepida asimilacioacuten y aumento de biomasa asociado se podriacutea traducir en un

aumento de la biodegradacioacuten (Ye et al 1996) Lee et al (2003) propusieron y

comprobaron que el piruvato potenciaba el crecimiento de la cepa degradadora


20

Pseudomonas putida lo que se tradujo en un aumento de la tasa de biodegradacioacuten de

naftaleno Chen amp Aitken (1999) han comprobado que el salicilato induce la siacutentesis de

enzimas cataboacutelicas y por tanto su adicioacuten favorece la metabolizacioacuten de HAP siempre

que su concentracioacuten en el medio no supere una concentracioacuten liacutemite inhibitoria Wong et al

(2000) observoacute que la adicioacuten de glucosa favoreciacutea el crecimiento total de la poblacioacuten pero

las tasas de biodegradacioacuten fueron significativamente menores Estos resultados se deben

a que la cepa o consorcio degradador es capaz de mineralizar un HAP como uacutenica fuente de

carbono y la asimilacioacuten de glucosa inhibe la siacutentesis de enzimas implicadas en la

degradacioacuten del contaminante Es necesario valorar en cada caso de estudio coacutemo afecta la

adicioacuten de una nueva fuente de carbono ya que en el caso de bacterias no pre-adaptadas a

degradar HAP es posible que sus efectos sean positivos (Wong et al 2000) pero en

poblaciones microbianas histoacutericamente adaptadas a substratos contaminados la adicioacuten de

glucosa puede favorecer el crecimiento de microorganismos heteroacutetrofos no degradadores

Importancia y efecto de los surfactantes en la biodegradacioacuten de HAP

La baja solubilidad de los HAP y de la mayoriacutea de los compuestos del petroacuteleo limita la

capacidad de los microorganismos para acceder y degradar los compuestos contaminantes

Los surfactantes son tensioactivos que actuacutean disminuyendo la tensioacuten superficial del agua

para facilitar la disolucioacuten de los HAP en la fase acuosa En algunos estudios (Bautista et al

2009) se ha demostrado que el uso de surfactantes en procesos de biodegradacioacuten es

necesario para solubilizar los HAP Muchas bacterias degradadoras de HAP han

desarrollado la capacidad de generar biosurfactantes (surfactantes de origen microbiano)

como parte de su superficie celular o como moleacuteculas liberadas extracelularmente (Fiechter

1992) Algunas de estas especies pertenecen a los geacuteneros Pseudomonas (P aeruginosa

P fluorescens) Rhodococcus Mycobacterium Lactobacillus Acinetobacter o

Sphingomonas Los biosurfactantes se clasifican en funcioacuten de su masa molecular en

biosurfactantes de bajo peso molecular como glicoliacutepidos o lipopeacuteptidos y de alto peso

molecular que incluyen moleacuteculas maacutes complejas como polisacaacuteridos anfipaacuteticos proteiacutenas

lipopolisacaacuteridos y lipoproteiacutenas Se han observado resultados muy contradictorios en

cuanto a sus efectos sobre las tasas de biodegradacioacuten por un lado positivos (Jing et al

2007) pero tambieacuten inhibitorios (Laha amp Luthy 1991) Los biosurfactantes de alto peso

molecular son eficaces en la estabilizacioacuten de emulsiones de aceite en agua mientras que

los de bajo peso molecular son maacutes eficaces en la disminucioacuten de la tensioacuten interficial y

superficial (Rosenberg amp Ron 1999) En el caso de los surfactantes sinteacuteticos su eficacia

estaacute determinada por sus propiedades de carga (no ioacutenicos anioacutenicos o catioacutenicos) su

balance hidrofiacutelico-lipofiacutelico y su concentracioacuten micelar criacutetica (CMC) concentracioacuten a la cual


21

la tensioacuten superficial es miacutenima y los monoacutemeros de surfactante se agregan formando

micelas Sin embargo algunos surfactantes pueden inhibir la mineralizacioacuten de los HAP por

cuestiones como la toxicidad del medio derivada de una elevada concentracioacuten de

surfactantes la cual resulta toacutexica para los microorganismos (ej Tergitol NP-10) o porque

al solubilizarse los HAP por accioacuten del surfactante aumenta toxicidad del medio (Liu et al

2001) En algunos casos ademaacutes los surfactantes no son biodegradables (Bautista et al

2009) De hecho Bautista et al (2009) comprobaron que el surfactante no ioacutenico Tergitol

NP-10 es un surfactante no biodegradable y toacutexico para los microorganismos en

comparacioacuten con el Tween-80 Dada la amplia variabilidad de resultados referentes a los

surfactantes es importante la eleccioacuten correcta en cada proceso considerando el tipo de

contaminante a eliminar y los microorganismos presentes en el medio

Biodegradacioacuten bacteriana cepas y consorcios microbianos degradadores de HAP

Son muchas las especies bacterianas descritas con capacidad degradadora de HAP la

mayoriacutea de ellas aisladas de aguas sedimentos y suelos previamente contaminados con

hidrocarburos La biodegradacioacuten de HAP de bajo peso molecular como el naftaleno

fenantreno y antraceno ha sido ampliamente estudiada Sin embargo son escasos los

estudios realizados sobre la biodegradacioacuten de HAP de alto peso molecular como el pireno

perileno acenafteno o fluoreno (Kanaly amp Harayama 2000) De acuerdo con Chauhan et al

(2008) especies de Pseudomonas y Ralstonia se relacionan en mayor medida con la

degradacioacuten de naftaleno y fenantreno Burkolderia y Stenotrophomonas con naftaleno

fenantreno y antraceno y otras especies pertenecientes a los geacuteneros Rhodococcus

Sphingomonas y Mycobacterium con HAP de mayor peso molecular como fluoranteno

benzo[a]pireno pireno benzo[b]fluoranteno Las especies bacterianas degradadoras

pertenecen a grupos filogeneacuteticos muy diversos y en muchos casos taxonoacutemicamente

alejados Trabajos previos (Vintildeas et al 2005 Molina et al 2009 Gonzaacutelez et al 2011)

muestran una gran parte de las bacterias degradadoras pertenecen al phylum

Proteobacteria en mayor proporcioacuten a las clases α- Proteobacteria (Sphingomonas

Bradyrizobium Nitrobacteria Balneimonas) y γ- (Pseudomonas Stenotrhophomonas

Enterobacter Pantoea Acinetobacter o Psychrobacter) Tambieacuten se han aislado especies

pertenecientes a la clase β- Proteobacterias (Ralstonia) y a los phylum Actinobacteria

(Microbacterium sp Rhodococcus sp) Firmicutes (Bacillus subtillis) y Bacteroidetes

(Flexibacter) aunque eacutestas en menor frecuencia Muchos de los trabajos de degradacioacuten

bacteriana se han realizado con cepas individuales (Grimberg et al 1996 Das amp Mukherjee

2006) extraiacutedas de suelos contaminados o bien con consorcios artificiales formados por

varias cepas degradadoras (Ghazali et al 2004) siendo muy pocos aquellos en los que se


22

ha utilizado un consorcio bacteriano natural extraiacutedo directamente de un suelo Bautista et al

(2009) sentildeala que la capacidad de degradacioacuten de un consorcio artificial es mayor que la de

las cepas individuales Seguacuten algunos autores (Fritsche 1985 Mueller et al 1997) la mejor

eficiencia de degradacioacuten de un consorcio es debido a que la diversidad de especies permite

que cada una tenga un papel en el proceso de biodegradacioacuten facilitando la degradacioacuten de

HAP gracias al cometabolismo establecido entre las especies implicadas

Existe una importante controversia referente a la capacidad degradadora que

presentan los consorcios naturales ya que se ha observado que ciertos consorcios

extraiacutedos de zonas no contaminadas con HAP son capaces de metabolizar dichos

compuestos (Tian et al 2008 Couling et al 2010) Seguacuten estos autores es una

caracteriacutestica general presente en algunas comunidades microbianas que se expresa ante

una determinada perturbacioacuten Sin embargo Barkay amp Pritchart (1988) exponen que es una

caracteriacutestica que soacutelo estaacute presente en comunidades previamente expuestas y por lo tanto

preadaptadas como consecuencia de presiones selectivas Algunos autores (Jhonsen et al

2005) subrayan que la capacidad cataboacutelica se propaga faacutecilmente entre bacterias de un

mismo suelo contaminado como resultado de la transferencia horizontal de genes (ej

conjugacioacuten y transformacioacuten) Esto facilitariacutea auacuten maacutes que una comunidad no preadaptada

pueda hacer frente a una perturbacioacuten

Teacutecnicas de biorremediacioacuten

El objetivo uacuteltimo de la biorremediacioacuten es que el proceso de biodegradacioacuten se desarrolle

de la forma maacutes eficaz posible y por eso muchos trabajos se centran en la optimizacioacuten del

proceso mediante el desarrollo de teacutecnicas que aumenten la eficacia del mismo Teacutecnicas

como la bioestimulacioacuten anteriormente mencionada se centran en aumentar la capacidad

degradadora de la comunidad autoacutectona bacteriana mediante la adicioacuten de nutrientes

(nitroacutegeno foacutesforo potasio) de forma que la falta de nutrientes no suponga una limitacioacuten

para el metabolismo microbiano y por consiguiente para la biorecuperacioacuten de la zona

perturbada Otras teacutecnicas se basan en el aumento de la poblacioacuten degradadora mediante la

adicioacuten de cepas o consorcios con capacidad reconocida para metabolizar un determinado

compuesto es lo que se conoce como bioaumento En algunos trabajos los resultados

derivados de la bioestimulacioacuten y el bioaumento han sido positivos (Mills et al 2004

Atagana 2006) pero en trabajos como Chen et al (2008) o Yu et al (2005) los efectos de

ambas teacutecnicas en el proceso fueron negativos o simplemente no tuvieron efecto Se tiene

que tener en cuenta que ambas teacutecnicas generan cambios en las comunidades autoacutectonas

que afectan y modifican las relaciones de competencia Estos cambios pueden ser auacuten maacutes


23

acusados en el caso del bioaumento pues la introduccioacuten de una comunidad foraacutenea puede

tener resultados difiacuteciles de predecir La atenuacioacuten natural es otra teacutecnica basada en la

mera actuacioacuten de la comunidad autoacutectona sin ninguacuten tipo de modificacioacuten de la comunidad

yo adicioacuten de sustancias Dowty et al (2001) entre otros defiende que cuando se trata de

restablecer el medio a las condiciones originales preservando la biodiversidad la

atenuacioacuten microbiana puede ser la mejor opcioacuten siempre que las poblaciones autoacutectonas

presenten capacidad degradadora

Resumen Objetivos

25

Objetivos

El objetivo general de la tesis es el conocimiento profundo de la biodegradacioacuten bacteriana

de hidrocarburos aromaacuteticos policiacuteclicos para la biorremediacioacuten y recuperacioacuten de medios

contaminados con estos compuestos toacutexicos El proyecto se ha centrado en la identificacioacuten

y conocimiento de la poblacioacuten bacteriana de consorcios procedentes de ambientes

(contaminados o no) y de su dinaacutemica ante determinadas condiciones durante el proceso de

biodegradacioacuten Para alcanzar este objetivo principal se fijaron objetivos especiacuteficos

desarrollados en cuatro capiacutetulos

1 Estudiar los factores abioacuteticos a fin de optimizar las condiciones de desarrollo en el

proceso de biodegradacioacuten de HAP en cultivos liacutequidos (capiacutetulo 1a) En un segundo

proyecto de optimizacioacuten el objetivo fue asemejar el proceso en laboratorio lo maacutes

posible a las condiciones naturales considerando los efectos derivados de la

interaccioacuten muacuteltiple de factores ambientales y bioloacutegicos (capiacutetulo 1b)

2 Analizar el efecto de la aplicacioacuten de distintos tipos de surfactantes (no ioacutenicos

biodegradables y no biodegradables) en la eficacia de degradacioacuten de HAP de un

consorcio bacteriano previamente adaptado (C2PL05) Asiacute mismo se quiso estudiar el

efecto del tipo de surfactante en la sucesioacuten y dinaacutemica del consorcio identificando los

microorganismos implicados a lo largo del proceso (capiacutetulo 2)

3 Estimar la capacidad degradadora de microcosmos inoculados con consorcios

procedentes de suelos con distinta historia de contaminacioacuten suelo croacutenicamente

contaminado (C2PL05) frente a un suelo procedente de un ambiente libre de

contaminacioacuten (BOS08) bajo condiciones climaacuteticas suaves y extremas Describir y

comparar las comunidades bacterianas que componen ambos consorcios (capiacutetulo 3)

4 Evaluar la eficacia de diversas teacutecnicas de biorremediacioacuten (atenuacioacuten natural

bioestimulacioacuten yo bioaumento) tanto en la eliminacioacuten del contaminante y la

toxicidad como en la capacidad de recuperacioacuten del ecosistema (capiacutetulo 4) Para el

desarrollo de este objetivo se llevoacute a cabo una simulacioacuten a pequentildea escala

(microcosmos) de un proceso de biorremediacioacuten in situ de suelos naturales

contaminados con creosota

Resumen Listado de manuscritos

27

Listado de manuscritos

Los capiacutetulos que integran este proyecto doctoral han sido redactados en ingleacutes para su

publicacioacuten en revistas cientiacuteficas de aacutembito internacional Por ello se presentan los

manuscritos originales de dichos artiacuteculos A continuacioacuten se detalla la traduccioacuten del tiacutetulo

los nombres de los coautores y el estado de publicacioacuten de los manuscritos

Capiacutetulo 1a Simarro R Gonzaacutelez N Bautista LF Sanz R y Molina MC

Optimisation of key abiotic factors of PAH (naphthalene phenanthrene

and anthracene) biodegradation process by a bacterial consortium

Water Air and Soil Pollution (2011) 217 365-374

Capiacutetulo 1b Simarro R Gonzaacutelez N Bautista LF y Molina MC

Evaluation of the influence of multiple environmental factors on the

biodegradation of dibenzofuran phenanthrene and pyrene by a bacterial

consortium using an orthogonal experimental design

Water Air and Soil Pollution (Aceptado febrero 2012)

Capiacutetulo 2 Gonzaacutelez N Simarro R Molina MC Bautista LF Delgado L y Villa

JA

Effect of surfactants on PAH biodegradation by a bacterial consortium and

on the dynamics of the bacterial community during the process

Bioresource Technology (2011) 102 9438-9446

Capiacutetulo 3 - Simarro R Gonzaacutelez N Bautista LF y Molina MC

High molecular weight PAH biodegradation by a wood degrading

consortium at low temperatures

FEMS Microbiology Ecology (Subscrito Diciembre 2011 En revisioacuten)


28

Capiacutetulo 4 - Simarro R Gonzaacutelez N Bautista LF Molina MC Peacuterez L y Peacuterez

M

Assessment the efficient of bioremediation techniques (biostimulation

bioaugmentation and natural attenuation) in a creosote polluted soil

change in bacterial community

Manuscrito ineacutedito

Resumen Siacutentesis de capiacutetulos

29

Siacutentesis de capiacutetulos

La presente tesis doctoral se desarrolla dentro de un proyecto de investigacioacuten basado en la

biorremediacioacuten de suelos contaminados con hidrocarburos aromaacuteticos policiacuteclicos y

sustancias contaminantes que los contengan Este proyecto se esta llevando a cabo desde

hace seis antildeos por el grupo de Biorremediacioacuten del Departamento de Biologiacutea y Geologiacutea de

la Universidad Rey Juan Carlos Los artiacuteculos citados anteriormente componen los cuatro

capiacutetulos que se desarrollan en el cuerpo de la tesis

Anteriormente al desarrollo de los experimentos que componen los capiacutetuos de la

presente tesis se realizoacute un trabajo (Molina et al 2009) en el que se estudioacute la capacidad

de degradacioacuten de un consorcio bacteriano extraiacutedo de un suelo croacutenicamente contaminado

y se describioacute su poblacioacuten bacteriana mediante teacutecnicas dependientes e independientes de

cultivo El consorcio fue capaz de degradar los HAP (naftaleno fenantreno y antraceno) en

maacutes de un 98 en un periodo de 18 diacuteas y ademaacutes eliminoacute completamente la toxicidad del

medio en 41 diacuteas La identificacioacuten molecular permitioacute aislar e identificar 5 cepas bacteriana

(DIC-1 a DIC-6 DIC Degrading isolated Culture) todas γ-Proteobacterias pertenecientes a

los geacuteneros Enterobacter Pseudomonas y Stenotrophomonas (maacutes detalles en Molina et al

2009) Ademaacutes el anaacutelisis de la poblacioacuten mediante electroforesis en un gel con gradiente

desnaturalizante (DGGE Denaturing Gradient Gel Electrophoresis) confirmoacute que estos tres

geacuteneros eran dominantes La alta eficacia en la degradacioacuten de los HAP y la escasa

biodiversidad tiacutepica de suelos sometidos a elevadas concentraciones de contaminantes

durante largos peridos de tiempo indican que el consorcio C2PL05 estaacute totalmente

adaptado a la degradacioacuten de HAP

En el capiacutetulo 1 se optimizoacute el proceso de biodegradacioacuten a traveacutes de dos disentildeos

experimentales diferentes que se recogen en los subcapiacutetulos 1a y 1b En el capiacutetulo 1a

se evaluacutea y mejora la influencia de varios factores ambientales como la relacioacuten molar de

CNP la fuente de nitroacutegeno fuente y concentracioacuten de hierro pH y fuente de carbono El

anaacutelisis estadiacutestico de las tasas de degradacioacuten (Kb) y los incrementos de la densidad celular

indicoacute que todos los factores fueron significativamente influyentes en la Kb Esto permitioacute

establecer un valor oacuteptimo de estos factores y descartar en los siguientes ensayos aquellos

paraacutemetros como el pH cuyo valor oacuteptimo (pH 7) seguacuten nuestros resultados coincidiacutea con

otros estudios y estaba claramente definido en la bibliografiacutea A partir de los resultados de

esta primera parte se realizoacute un segundo ensayo de optimizacioacuten ortogonal multifactorial

(capiacutetulo 1b) que permitioacute la optimizacioacuten de 8 factores de forma conjunta La concentracioacuten

de surfactante y de inoacuteculo bacteriano son dos factores que no fueron incluiacutedos en el

anterior anaacutelisis pero a traveacutes de la bibliografiacutea se consideraron factores importantes en la


30

biodegradacioacuten de HAP Los resultados confirmaron que la temperatura la fuente de

carbono y la concentracioacuten de inoacuteculo fueron factores significativos en el incremento de la

densidad celular pero soacutelo la fuente de carbono influyoacute significativamente el porcentaje total

de degrad acioacuten Estos resultados (capiacutetulo 1) permitieron conocer cuaacuteles son las

condiciones oacuteptimas del cultivo para una degradacioacuten maacutes eficaz de HAP con el consorcio

bacteriano C2PL05

El uso de surfactantes en la biodegradacioacuten de HAP puede optimizar la eficacia del

proceso y en algunos casos su uso es imprescindible Sin embargo su utilizacioacuten implica

un elevado coste y en ocasiones pueden tener efectos negativos dependiendo de la

concentracioacuten y tipo de surfactante En el capiacutetulo 2 se evaluacutea el efecto de dos

surfactantes no ioacutenicos Tergitol NP-10 (no biodegradable) y Tween-80 (biodegradable) en

la capacidad degradadora del consorcio microbiano C2PL05 teniendo en cuenta la

velocidad de degradacioacuten de los HAP y la dinaacutemica de la poblacioacuten microbiana durante el

proceso (teacutecnicas cultivo-dependientes e independientes) La degradacioacuten bacteriana de

los HAP y la reduccioacuten de la toxicidad del medio fue significativamente mejor con el

surfactante no ioacutenico y biodegradable Tween-80 Ademaacutes el tipo de surfactante utilizado

para optimizar la biodegradacioacuten de HAP afectoacute significativamente a la dinaacutemica de la

comunidad bacteriana del consorcio siendo especies pertenecientes a los geacuteneros

Pseudomonas Sphingomonas Sphingobium y Agromonas responsables de estas

diferencias Teoacutericamente la uacutenica funcioacuten del surfactante en el proceso de

biodegradacioacuten es aumentar la solubilidad de los HAP pero sin embargo en este capiacutetulo

se ha puesto de manifiesto que modifican notablemente las poblaciones bacterianas y la

sucesioacuten de especies pudiendo afectar a la eficacia del proceso Un estudio previo que

desemboque en la eleccioacuten del surfactante maacutes adecuado asiacute como su concentracioacuten

favorece la efiacacia de la biorremediacioacuten

El capiacutetulo 3 se centra en el estudio de la capacidad degradadora de los

microorganismos El principal objetivo es comprobar si la capacidad de degradacioacuten se

adquiere necesariamente tras un periodo de exposicioacuten a un contaminante o si bien es una

caracteriacutestica intriacutensecamente presente en algunas bacterias Se considera que la

temperatura es uno de los factores maacutes influyentes en el proceso de biodegradacioacuten de

manera que a temperaturas friacuteas (lt15 ordmC) o extremas (lt5 ordmC) se dificulta el proceso porque

afecta a la solubilidad de los HAP y al metabolismo microbiano Sin embargo existen

especies que toleran o estaacuten adaptadas a las bajas temperaturas y que ademaacutes pueden

degradar HAP En este capiacutetulo se avaluoacute la capacidad degradadora de un consorcio

preadaptado a HAP (C2PL05) frente a un consorcio extraiacutedo de una zona priacutestina rica en

madera en descomposicioacuten (BOS08) Al mismo tiempo se simularon dos ambientes de


31

biodegradacioacuten uno a temperaturas suaves comprendidas entre los 15 ordmC-25 ordmC y otro maacutes

extremo con temperaturas entre los 5 ordmC-15 ordmC todo ello en un sustrato soacutelido (suelo) con

objeto de ajustarnos a las condiciones naturales Sin duda el resultado maacutes significativo fue

que el consorcio BOS08 no adaptado a la degradacioacuten de HAP fue capaz de degradar

eficazmente e incluso mejor que el consorcio C2PL05 cuando las temperaturas fueron bajas

Ademaacutes se observoacute que aunque hubo geacuteneros exclusivos de cada consorcio (Ralstonia

Bacillus) otros fueron comunes en ambos (Microbacterium Acinetobacter Pseudomonas)

Los resultados obtenidos confirman la hipoacutetesis de que la capacidad de degradacioacuten estaacute

presente de forma intriacutenseca en algunas especies y no depende de una pre-exposicioacuten al

contaminante

En la biorremediacioacuten de un suelo contaminado con HAP es muy importante tener en

cuenta la respuesta de la poblacioacuten bacteriana autoacutectona del suelo frente a un episodio de

contaminacioacuten En el capiacutetulo 4 se estudioacute el comportamiento de la comunidad bacteriana

de un suelo previamente no contaminado cuando es perturbado con creosota La

biorremediacioacuten in situ es un proceso maacutes complejo que cuando se estudia bajo condiciones

controladas de laboratorio ya que hay factores como la escasez de nutrientes o las bajas

temperaturas que dificultan el proceso (capiacutetulos 1 y 3) Sin embargo la aplicacioacuten de

tratamientos in situ como la bioestimulacioacuten o el bioaumento pueden mejorar la eficacia de la

biorremediacioacuten En este capiacutetulo se determinoacute la respuesta de la comunidad bacteriana

frente a la bioestimulacioacuten el bioaumento o a la atenuacioacuten natural evaluando el porcentaje

de degradacioacuten de creosota y los HAP que la componen la reduccioacuten de la toxicidad y al

mismo tiempo estudiando los cambios poblacionales El criterio para la eleccioacuten de la

teacutecnica maacutes eficaz se determinoacute en funcioacuten de la eficacia en la degradacioacuten y en la

reduccioacuten de la toxicidad Los resultados mostraron que aunque la creosota se redujo

considerablemente y sin diferencias significativas entre tratamientos la toxicidad del medio

permanecioacute alta durante todo el proceso La ausencia de diferencias significativas entre

tratamientos en cuanto a la eliminacioacuten de creosta se refiere indica que la comunidad

autoacutectona del suelo tiene mcroorgasnimos con capacidad degradadora aunque previamente

no hayan estado expuestos a HAP Las bajas temperaturas a las que se desarrolloacute el

experimento fue la causa de que la toxicidad del medio no se redujera Cabe destacar la

importancia de las identificaciones mediante teacutecnicas no cultivables de especies

pertenecientes a los geacuteneros Balneimonas y Pantoea previamente no descritas en procesos

de biodegradacioacuten de creosota o HAP

Resumen Metodologiacutea general

33

Metodologiacutea general

Todos los materiales y meacutetodos estaacuten descritos y con sus respectivas refrencias en cada

uno de los capiacutetulos que se indican a continuacioacuten Sin embargo en algunos casos y dado

que la publicacioacuten de los datos en muchos casos obliga a ajustarse a los formatos de cada

revista especializada algunos meacutetodos no se han explicado en detalle en los capiacutetulos Este

apartado ldquoMetodologiacutea generalrdquo tiene como objetivo profundizar en el fundamento de

algunos de los meacutetodos utilizados durante el desarrollo de este proyecto

Preparacioacuten de consorcios bacterianos

El consorcio bacteriano C2PL05 fue utilizado en los experimentos de los capiacutetulos que

componen esta tesis como consorcio degradador de HAP Este consorcio fue extraiacutedo de un

suelo croacutenicamente contaminado con HAP (Figura 2A) de una refineriacutea de petroacuteleo situada

en Puertollano Ciudad Real (Espantildea) Su capacidad degradadora se verificoacute en un cultivo

semicontinuo en matraces Erlenmeyer de 100 ml que conteniacutean 50 ml BHB con Tween-80

(1 vv) y naftaleno fenantreno (05 gmiddotlminus1) y antraceno (005 gmiddotlminus1) realizando refrescos del

medio cada 15 diacuteas

Cuando se indique (capiacutetulo 3) se preparoacute un consorcio de un suelo procedente de un

bosque (43ordm 4175acuteN 8ordm 0683acuteO Frgas do Eume Galicia Espantildea Figura 2B) totalmente

libre de contaminacioacuten por HAP al que se denominoacute BOS08 El suelo se obtuvo de la parte

maacutes superficial por lo que teniacutea un alto contenido en materia orgaacutenica y restos de madera

muerta

Figura 2 Suelo contaminado procedente de la refineriacutea (A) y suelo

procedente de bosque (B) de los cuales se extrajeron los consorcios

C2PL05 y BOS08 respectivamente

A B


34

Para la extraccioacuten de ambos consorcios bacterianos 1 g de suelo se resuspendioacute en

10 ml de solucioacuten salina PBS (pH 70) y se mantuvo en agitacioacuten constante a 150 rpm en

oscuridad y a 25ordmC durante 24 horas Posteriormente se formoacute un cultivo madre de cada

consorcio en 50 ml de BHB con la mezcla de HAP que se fuera a utilizar en el experimento

tween-80 (1 vv) como surfactante y 15 ml del extracto de cada consorcio Los cultivos se

incubaron en un agitador orbital a 150 rpm y 25ordmC hasta que alcanzara la fase exponencial

En este momento se inoculaba la cantidad de cultivo madre necesario en los microcosmos

de los experimentos en funcioacuten de la concentracioacuten de inoacuteculo deseada en los mismos

Disentildeos experimentales

En este apartado se explica el disentildeo y la composicioacuten de los experimentos que conforman

los capiacutetulos de esta tesis para facilitar su comprensioacuten Generalizando los capiacutetulos 1 (1a y

1b) y 2 tratan de optimizar el medio nutritivo suministrado al consorcio C2PL05 el tipo y

concentracioacuten de surfactante y otros factores ambientales para lo cual los microcosmos

eran cultivos liacutequidos incubados en Erlenmeyers en un agitador orbital Los capiacutetulos 3 y 4

se centran en el estudio de la biodegradacioacuten de HAP en sustrato soacutelido (arena de riacuteo y

suelo natural respectivamente) para reproducir en la medida de los posible las condiciones

naturales

En el capiacutetulo 1a se optimizaron 6 factores (3 posibles valores cada factor) de forma

individual y consecutiva En total se desarrollaron 18 tratamientos en cultivos liacutequidos (3

reacuteplicas) incubados en un agitador orbital a 150 rpm 25 ordmC y oscuridad (Figura 3) durante

168 horas En el capiacutetulo 1b el disentildeo ortogonal L18 (37) (21) permitioacute mediante el desarrollo

de 18 tratamientos (3 reacuteplicas cada uno) la optimizacioacuten de 8 factores 7 de ellos con 3

posibles valores (37) y un factor con dos valores (21) Un total de 54 cultivos se incubaron

durante 159 horas en las mismas condiciones que en el capitulo 1a variando la temperatura

seguacuten las necesidades del tratamiento En la Figura 4 y 5 se muestran los disentildeos

experimentales correspondientes a los capiacutetulos 1a y 1b respectivamente


35

Figura 3 Cultivos liacutequidos incubados en un agitador orbital

Optimizacioacuten

CNP

BHB tween-80

C2PL05Naftaleno fenantreno

antraceno

X 3

100101

1002116

100505

Optimizacioacuten

fuente de N

BHB tween-80


antraceno

X 3

NaNO3

NH4NO3

(NH4)2SO3

Optimizacioacuten

fuente de Fe

BHB tween-80


antraceno

X 3

FeCl3

Fe(NO3)3

Fe2(SO4)3

Optimizacioacuten

[Fe]

BHB tween-80


antraceno

X 3

005 mM

01 mM

02 mM

Optimizacioacuten

pH

BHB tween-80


antraceno

X 3

50

70

80

Optimizacioacuten

fuente de C

BHB tween-80

C2PL05

Naftaleno fenantreno

antraceno y glucosa (20 80 100)

X 3

HAP

HAPglucosa (5050)

Glucosa

2ordm 3ordm

4ordm 5ordm 6ordm

Figure 4 Disentildeo experimental correspondiente al experimento que conforma el capiacutetulo 1a


36

Tordf

Optimizacioacuten CNP

OptimizacioacutenFuente N

OptimizacioacutenFuente Fe

Optimizacioacuten[Fe]

Optimizacioacuten[Tween-80]

Optimizacioacutendilucioacuten inoacuteculo

Optimizacioacutenfuente de C

20ordmC25ordmC30ordmC

1001011002116100505

NaNO3

NH4NO3

(NH4)2SO3

FeCl3Fe(NO3)3

Fe2(SO4)3

005 mM01 mM02 mM

CMC20 CMC

10-1

10-2

10-3

0100505020100

18 tratamientos

X 3

C2PL05Antraceno dibenzofurano pireno

BHB (modificado seguacuten tratamiento)

Figura 5 Disentildeo experimental correspondiente a al experimento que conforma el capiacutetulo 1b

En el capiacutetulo 2 se estudioacute la capacidad de degradacioacuten y la evolucioacuten del consorcio

C2PL05 en dos tratamientos (cada uno en triplicado) uno adicionado con Tween-80 y otro

con Tergitol NP-10 (1 vv) como surfactantes En total 6 cultivos liacutequidos se incubaron a

150 rpm 25 ordmC y oscuridad durante 45 diacuteas como se muestra en la Figura 3 El disentildeo

experimental de este capiacutetulo se resume graacuteficamente en la Figura 6

Tratamiento 1con Tween-80

Tratamiento 2con Tergitol NP-10

C2PL05BHB-Tergitol NP-10 (1)Naftaleno fenantreno antraceno

X 3

X 3

C2PL05BHB-Tween-80 (1)Naftaleno fenantreno antraceno

Figura 6 Disentildeo experimental correspondiente al experimento que conforma

el capiacutetulo 2


37

El capiacutetulo 3 se desarrolloacute en microcosmos con 90 g de arena de riacuteo esterilizada

(Figura 7) e incinerada en una mufla a 300 ordmC para eliminar cualquier tipo de

microorganismos o materia orgaacutenica que pudiera contener Se realizaron 4 tratamientos

distintos en funcioacuten de la temperatura de incubacioacuten (5-15 ordmC o 15-25 ordmC) y del consorcio

inoculado (C2PL05 o BOS08) cada tratamiento con tres reacuteplicas para cada uno de los 5

tiempos de muestreo lo que supuso un total de 60 microcosmos (Figura 8) Los nutrientes

se suministraron antildeadiendo 18 ml de medio BHB que proporcionaba una humedad relativa

del 60 y que ademaacutes conteniacutea Tween-80 (1 vv) Los microcosmos se inocularon con

35 ml de un cultivo madre del consorcio C2PL05 oacute BOS08 y se incubaron en caacutemaras bajo

condiciones controladas de temperatura (seguacuten tratamiento) humedad (60 constante) y

luz (16 horas de luz8 horas oscuridad)

Figura 7 Microcosmos del experimento para el capiacutetulo 3 en caacutemara de crecimiento


38

Tratamiento 1

Tratamiento 2

Tratamiento 3

Tratamiento 4

C2PL0515-25ordmCBHB oacuteptimoNaftaleno fenantreno antracenopireno y perileno

C2PL055-15ordmCBHB oacuteptimoNaftaleno fenantreno antraceno pireno y perileno

BOS0815-25ordmCBHB oacuteptimoNaftaleno fenantreno antraceno pireno y perileno


Arena esterilizada +




X 3

X 3

X 3

X 3

X 5 tiempos

X 5 tiempos

X 5 tiempos

X 5 tiempos

TOTAL = 60 MICROCOSMOS

Figura 8 Disentildeo experimental correspondiente al experimento que conforma el capiacutetulo 3

El experimento que compone el capiacutetulo 4 de biorremediacioacuten in situ se desarrolloacute

bajo condiciones ambientales externas en una zona del campus preparada para ello Como

sustrato se utilizoacute suelo natural extraiacutedo de los primeros 20 cm y tamizado con una maya lt

2 mm El sustrato se depositoacute en bandejas de plaacutestico para evitar que el agente

contaminante se filtrara al suelo y se protegioacute de la lluvia tal y como se observa en la Figura

9 Cada uno de los cinco tratamientos (control atenuacioacuten natural bioestimulacioacuten

bioaumento y bioestimulacioacuten junto con bioaumento) se hizo en duplicado para cada uno de

los 4 muestreos realizados durante los 8 meses de experimentacioacuten (octubre-junio) Cada

microcosmos (bandeja) conteniacutea 550 g de suelo humedecidos (40) con agua o BHB como

fuente de nutrientes en el caso de los tratamientos con bioestimulacioacuten Los tratamientos

bioaumentados se enriquecieron con 5 ml de un cultivo madre del consorcio C2PL05 Como

agente contaminante se utilizoacute creosota antildeadiendo 25 ml de una disolucioacuten de creosota en


39

n-hexano (25 g creosota por bandeja) a todos los tratamientos excepto al control Resumen

del disentildeo en la Figura 10

Figura 9 Experimento para el capiacutetulo 4 bajo condiciones ambientales

externas en el Campus de la Universidad Rey Juan Carlos Moacutestoles

Tratamiento 1 Control

Tratamiento 2 Atenuacioacuten

natural

Tratamiento 3 Bioestimulacioacuten

Tratamiento 4 Bioaumento


y Bioaumento

Suelo sin contaminar X 4 tiempos

CreosotaH2O-Tween-80 X 4 tiempos

CreosotaBHB oacuteptimo-Tween-80 X 4 tiempos

CreosotaH20 ndash Tween-80 X 4 tiemposC2PL05

CreosotaBHB oacuteptimo-Tween-80 X 4 tiemposC2PL05

Suelo natural +X 2

Suelo natural +X 2

Suelo natural +X 2

Suelo natural +X 2

Suelo natural +X 2




40

Anaacutelisis fiacutesico-quiacutemicos

La caracterizacioacuten del suelo contaminado del cual se extrajo el consorcio C2PL05 asiacute como

la explicacioacuten de las teacutecnicas y metodologiacuteas empleadas se detallan en Molina et al (2009)

No obstante en la Tabla 1 se presentan las propiedades fiacutesico-quiacutemicas de dicho suelo

contaminado

Tabla 1 Propiedades fisico-quiacutemicas y bioloacutegicas del suelo contaminado con HAP

Propiedades Unidades Media plusmn ES

Tamantildeo medio de partiacutecula μm3 291 plusmn 6 Composicioacuten (arenaslimosarcillas) v 291009000 plusmn 03603600

pH - 77 plusmn 01

Conductividad μSmiddotcm-1 74 plusmn 22

WHCa v 33 plusmn 7

(NO3)- μgmiddotKg-1 40 plusmn 37


(NH4)+ μgmiddotKg-1 155 plusmn 125

(PO4)3- μgmiddotKg-1 47 plusmn 6

Carbono total v 96 plusmn 21

TOCb (tratamiento aacutecido) v 51 plusmn 04

MPNc (heteroacutetrofos) x104 ceacutelulasmiddotg-1 97 plusmn 12

MPNc (degradador de HAP) x103 ceacutelulasmiddotg-1 93 plusmn 19

Toxicity EC50d gmiddot100ml-1 144 plusmn 80

Hidrocarburos extraiacutedos w 92 plusmn 18

a Capacidad de campo del agua (WHC Water holding capacity) maacutexima cantidad de agua que

puede contener un suelo b Carbono orgaacutenico total (TOC total organic carbon) c Nuacutemero maacutes

probable (MPN most probably number) teacutecnica cultivo-dependiente que estima el nuacutemero de

ceacutelulas capaces de crecer con una determinada fuente de carbono (ver apartado Anaacutelisis

bioloacutegicos) d EC50 es una medida de la eficacia de una determinada sustancia o de su toxicidad

y representa la cantidad de un compuesto necesaria para disminuir al 50 una funcioacuten En

nuestro caso indica la toxicidad del medio que disminuye a la mitad la actividad microbiana del

consorcio

La metodologiacutea utilizada para la extraccioacuten de HAP (capiacutetulos 1 2 y 3) y creosota

(capiacutetulo 4) se detalla en el apartado de material y meacutetodos de los capiacutetulos

correspondientes La cromatografiacutea liacutequida de alto rendimiento (HPLC high-performance

liquid chromatography) fue la teacutecnica utilizada para el anaacutelisis de los HAP en los capiacutetulos 1

y 2 siguiendo el protocolo descrito por Bautista et al (2009) El equipo de anaacutelisis HPLC

(Prestar 230 Varian Palo Alto CA USA) esta compuesto por una columna C18 en fase

reversa Luna C18 (2) (75 cm longitud x 46 mm diaacutemetro interno y 3 μm tamantildeo de partiacutecula

Phenomenex Torrance CA USA) y conectado a una matriz de fotodiodos de UVVis


41

(ultravioletavisible) y un detector de fluorescencia La fase moacutevil utilizada se compone de un

gradiente acetonitriloagua programado como se detalla a continuacioacuten gradiente isocraacutetico

6040 (0-2 min) gradiente de 7525 (2-14 min) gradiente isocraacutetico 7525 (14-15 min)

gradiente de 1000 (15-16 min) El volumen de inyeccioacuten de muestra es de 10 μl y el flujo de

elusioacuten en la columna de 08 mlmiddotmin-1 La deteccioacuten de los HAP se realiza a 254 nm El

posterior tratamiento de los datos se detalla en los respectivos capiacutetulos

El meacutetodo para la deteccioacuten de HAP en el capiacutetulo 3 y de creosota en el capitulo 4 fue

la cromatografiacutea de gases (GC Gas Chromatography) utilizando un cromatoacutegrafo de gases

(system Varian 3900 Palo Alto CA USA) con un detector con ionizador de llama (FID

Flame Ionization Detector) La metodologiacutea protocolo y posterior tratamiento de los datos se

detallan en el material y meacutetodos de los respectivos capiacutetulos

Anaacutelisis bioloacutegicos

La densidad celular es una teacutecnica utilizada en todos los ensayos que componen esta tesis y

por tanto la metodologiacutea y el posterior tratamiento de los datos estaacuten detalladamente

descritos en todos los manuscritos que conforman los capiacutetulos de la tesis

Las teacutecnicas cultivo dependientes desarrolladas en los ensayos han sido el NMP

descrito en los capiacutetulos 2 3 y 4 y el aislamiento de colonias aplicando la metodologiacutea

empleada por Molina et al (2009) y descrita en los capiacutetulos 2 y 3

Teacutecnicas moleculares

Extraccioacuten y amplificacioacuten de ADN

La extraccioacuten de ADN de muestras de cultivos liacutequidos y muestras de biomasa de una

colonia aislada (capiacutetulos 2 y 3) se realizoacute usando el kit de extraccioacuten fiacutesico-quiacutemico de ADN

bacteriano Microbial DNA isolation Kit (Laboratorios MoBio Solano Beach CA USA) Para

la extraccioacuten de ADN total de muestras de suelo (capiacutetulos 3 y 4) se usoacute el kit de extraccioacuten

fiacutesico-quiacutemico Power Soil DNA kit (Laboratorios MoBio Solano Beach CA USA) siguiendo

en ambos casos el protocolo recomendado por el fabricante


42

Para la amplificacioacuten de las secuencias de ADN se utilizaron varias parejas de

cebadores en funcioacuten de la teacutecnica posterior de anaacutelisis del producto de PCR La

amplificacioacuten del ADN se realizoacute con el fin de identificar las secuencias de las cepas

aisladas o para un posterior anaacutelisis del ADN total de una muestra mediante electroforesis

en gel con gradiente desnaturalizante (DGGE denaturing gradient gel electrophoresis)

Cuando fue necesario reamplificar material geneacutetico procedente de una banda de DGGE la

pareja de primers utilizada no presentaba cola de GC (guanina-citosina) En la Tabla 2 se

describen las caracteriacutesticas de los cebadores y en la Figura 11 se detallan las condiciones

del programa correspondiente a cada pareja de cebadores

Tabla 2 Caracteriacutesticas de los cebadores utilizados para la amplificacioacuten de ADN por PCR

Cebador Secuencia 5acute--3acute Nordm de bases

Tordf hibridacioacuten

(ordmC)

Programa de PCR (Figura

Teacutecnica de anaacutelisis del producto de

16F27 AGAGTTTGATCMTGGCTCAG 20 55 I Purificacioacuten Secuenciacioacuten16R1488 CGGTTACCTTGTTACGACTTCAGG 24 55 I

16F341 CCTACGGGAGGCAGCAG 17 54 II DGGE Clonacioacuten Ecoli 16R907 CCGTCAATTCCTTTRAGTTT 20 54 II

16F338 CTCCTACGGGAGGCAGCAG 19 55 II DGGE Clonacioacuten Ecoli 16R518 CGTATTACCGCGGCTGCTGG 20 55 II

ITS1F CTTGGTCATTTAGAGGAAGTAA 20 54 III Presencia material geneacutetico ITS4 TCCTCCGCTTATTGATATGC 20 54 III

Primer con posibilidad de antildeadir una secuencia de 40 bases (5acute-CGC CCG CCG CGC CCC GCG

CCC GTC CCG CCG CCC CCG CCC G-3acute) rica en guanina (G) y citosina(C) unida al extremo 5acute- del

cebador necesaria para electroforesis en gel con gradiente desnaturalizantede


43

Figura 11 Condiciones de los programas de PCR I II y III a) Paso en el cual se procede a la

activacioacuten del principio activo de la polimerasa ExTaq-HS a una Tordf de activacioacuten de 94ordmC b) Tordf de

desnaturalizacioacuten c) Tordf de hibridacioacuten d) Tordf de polimerizacioacuten e) Tordf de extensioacuten f) Tordf final y de

conservacioacuten del producto de PCR

Desnata inicial Tordf desnatb

Tordfhibridc

Tordf pold Tordf exte

Tordf finalf

4 ordmC infin

95 ordmC 5 min

95 ordmC 1 min

54 ordmC 05 min

72 ordmC 15 min

72 ordmC 10 min

30 CICLOS

PROGRAMA PCR III


Tordfhibridc


Tordf finalf

4 ordmC infin

95 ordmC 9 min

94 ordmC 1 min

55 ordmC 1 min

72 ordmC 15 min

72 ordmC 5 min

30 CICLOS

PROGRAMA PCR II


Tordfhibridc


Tordf finalf

4 ordmC infin

94 ordmC 9 min

94 ordmC 1 min

55 ordmC 1 min

72 ordmC 15 min

72 ordmC 5 min

30 CICLOS

PROGRAMA PCR I


44

Electroforesis en gel con gradiente de desnaturalizacioacuten (DGGE) y clonacioacuten en

Escherichia coli

El estudio de la comunidad bacteriana se realizoacute mediante la teacutecnica DGGE ampliamente

descrita en los capiacutetulos 2 3 y 4 Las bandas maacutes predominantes fueron extraiacutedas del gel

eluiacutedas en de agua esterilizada y almacenadas a -20 ordmC para su posterior reamplificacioacuten y

clonacioacuten Las imaacutegenes de DGGE se analizaron graacutefica y estadiacutesticamente para diferenciar

entre las comunidades objeto de estudio y la influencia de los tratamientos en los cambios

de una comunidad El anaacutelisis graacutefico mediante el programa UN-Scan-It (v 60 Silk Scientific

US) permitioacute identificar el porcentaje de abundancia de cada banda con respecto de una

comunidad

La clonacioacuten en Ecoli se empleoacute debido a la imposiblidad de reamplificar el ADN

contenido en una banda cortada del gel de DGGE La metodologiacutea empleada para el

desarrollo de esta teacutecnica en los capiacutetulos 2 3 y 4 es la recomendada por el fabricante del

kit utilizado pGEM-T Easy Vector System II (Pomega)

Alineamiento de secuencias y anaacutelisis filogeneacuteticos

Las secuencias fueron editadas utilizando el programa Chromas Pro v142 que permite

ademaacutes visualizar y modificar posibles ambiguumledades en los nucleoacutetidos Las secuencias

fueron descargadas en las bases de datos disponibles (Genbank

(httpwwwncbinlmnihgovgenbank) y Ribosomal Sequence Data

(httprdpcmemsueduseqmatchseqmatch_introjsp)) a traveacutes de la opcioacuten BLAST con el

fin de aproximarnos a la identificacioacuten molecular de los organismos La secuencias fueron

alineadas utilizando el programa Bioedit v709 y posteriormente se elaboroacute una matriz de

datos a partir de la cual se establecieron las posibles relaciones filogeneacuteticos entre las

secuencias problema y aquellas descargadas de las bases de datos El programa utilizado a

tal efecto fue PAUP 40B10 (Swofford 2003)

Se utilizaron dos tipos de anaacutelisis para estimar las relaciones filogeneacuteticas y la

fiabilidad de las topologiacuteas obtenidas Por un lado se utilizoacute la Maacutexima Parsimonia estaacutendar

(Maxima Parsimonia de Fitch MP) un meacutetodo que elige el aacuterbol que requiere el menor

nuacutemero de cambios evolutivos para explicar las relaciones entre taxones a partir de la

informacioacuten generada por los sitios informativos el valor relativo de los diferentes caracteres

y de sus transformaciones Para evaluar la fiabilidad de las relaciones establecidas por

parsimonia se selecciono el meacutetodo Bootstraping (Felsenstein 1985) donde los caracteres


45

de las matrices se combinan al azar con las repeticiones necesarias considerando los

paraacutemetros establecidos en el anaacutelisis de parsimonia Asiacute el porcentanje con que aparece

un determinado grupo es una medida de la bondad de dicho grupo o rama Por otro lado la

diferencia entre secuencias (distancia geneacutetica) se computoacute en teacuterminos de nuacutemero de

nucleoacutetidos diferentes por sitio entre secuencias realizando un anaacutelisis de neighbour-joining

de acuerdo al algoritmo de Jukes amp Cantor (1969) Ambos tipos de anaacutelisis se llevaron a

cabo usando el software PAUP 40B10 (Swofford 2003)

Anaacutelisis estadiacutesiticos

Todos los capiacutetulos se disentildearon de forma que tras la finalizacioacuten del experimento los datos

pudieran ser tratados estadiacutesticamente El tratamiento estadiacutestico y los anaacutelisis realizados

con los datos de los ensayos estaacuten descritos en el apartado correspondiente de los

manuscritos que componen los artiacuteculos de la presente tesis Es necesario explicar

detalladamente el experimento que compone el capiacutetulo 1b disentildeado con un experimento

ortogonal del tipo L18 (37) (21) seleccionado del modulo de Statistica (Version 60) Este tipo

de disentildeo permitioacute hacer una combinacioacuten de muacuteltiples factores de forma reducida Es decir

un total de 18 experimentos representan todas las combinaciones posibles que se pueden

dar entre 8 factores siete de los cuales pueden tomar tres valores (37) diferentes (ej factor

Tordf valores 30ordmC 25ordmC y 20ordmC) y uno de ellos con dos valores (21) (ej factor concentracioacuten

de surfactante valores CMC y +20 CMC)

Para visualizar cambios en las comunidades microbianas (patrones univariantes) en

cada combinacioacuten de factores (ej tiempo tipo de consorcio temperatura) se realizoacute una

ordenacioacuten multivariante mediante un escalamiento multidimensional no-meacutetrico (MDS non-

parametric Multidimensional Scaling) usando el programa PRIMER (Clarke 1993) Este tipo

de anaacutelisis se realizoacute en los capiacutetulos 3 y 4 La ordenacioacuten se hizo sobre la base de la matriz

de dismilaridad de Bray-Curtis construida a partir de la transformacioacuten de los datos de

abundancia por la raiacutez cuarta para minimizar la influencia de los valores maacutes extremos

(Clarke 1993 Martiacuten Guirao 2007) Se usoacute el procedimiento SIMPER (Clarke 1993) para

identificar el porcentaje de contribucioacuten de cada banda a la disimilitud entre tratamientos

establecida en la matriz de Bray-Curtis Las bandas se consideraron influyentes cuando su

contribucioacuten se encontraba dentro de los primeros 70 (capiacutetulo 2) 60 (capiacutetulo 3) o 50

(capiacutetulo 4 Viejo 2009) del porcentaje medio de similaridaddisimilaridad acumulado entre y

dentro de la combinacioacuten de factores El criterio aplicado para escoger el porcentaje de

contribucioacuten limite se hizo en funcioacuten de la riqueza de bandas de forma que cuanto menor

fuera este paraacutemetro mayor el porcentaje liacutemite

Capiacutetulo

Publicado en Water Air amp Soil Pollution (2011) 217 365-374

Simarro R Gonzaacutelez N Bautista LF Sanz R amp Molina MC

Optimisation of key abiotic factors of PAH (naphthalene phenanthrene and

anthracene) biodegradation process by a bacterial consortium

Optimizacioacuten de los principales factores abioacuteticos de un proceso de biodegradacioacuten

de HAP (naftaleno fenantreno y antraceno) por un consorcio bacteriano

1a

Capiacutetulo 1a Optimisation of abiotic factors of PAH biodegradation process

49

Abstract

The aim of this work is to determine the optimum values for the biodegradation process of six

abiotic factors considered very influential in this process The optimization of a polycyclic

aromatic hydrocarbons (naphthalene phenanthrene and anthracene) biodegradation

process was carried out with a degrading bacterial consortium C2PL05 The optimized

factors were the molar ratio of carbonnitrogenphosphorus (CNP) the nitrogen source the

iron source the iron concentration the pH and the carbon source Each factor was optimized

applying three different treatments during 168 h analyzing cell density by spectrophotometric

absorbance at 600 nm and PAH depletion by HPLC To determine the optimum values of the

factors an analysis of variance (ANOVA) was performed using the cell density increments

and biotic degradation constants calculated for each treatment The most effective values of

each factor were a CNP molar ratio of 1002116 NaNO3 as nitrogen source Fe2(SO4)3 as

iron source using a concentration of 01 mmolmiddotl-1 a pH of 70 and a mixture of glucose and

PAH as carbon source Therefore high concentration of nutrients and soluble forms of

nitrogen and iron at neutral pH favour the biodegradation Also the addition of glucose to

PAH as carbon source increased the number of total microorganism and enhanced the PAH

biodegradation due to augmentation of PAH degrader microorganisms It is also important to

underline that the statistical treatment of data and the combined study of the increments of

the cell density and the biotic biodegradation constant has facilitated the accurate

interpretation of the optimization results For an optimum bioremediation process is very

important to perform these previous bioassays to decrease the process development time

and so the costs


51

Introduction

Polycyclic aromatic hydrocarbons (PAH) are persistent organic compounds with two or more

aromatic rings They are formed by incomplete combustion of fossil fuels and pyrolysis of

organic matter derived from human activities and as a result of natural events like forest fires

The toxic mutagenic and carcinogenic properties of PAH have concerned the Unites States

Environmental Protection Agency (US-EPA) proposing some of them as priority pollutants

(including naphthalene phenanthrene and anthracene) In addition the PAH solubility is very

low in aqueous medium (Luning Prak amp Pritchard 2002) affecting their degradation and

biomagnification within the ecosystems The microbial bioremediation removes or

immobilizes the pollutants reducing toxicity with a very low environmental impact Generally

microbial communities present in PAH contaminated soils are enriched by microorganisms

able to use them as only carbon source (Heitkamp amp Cerniglia 1988 Gallego et al 2007)

However this process can be affected by a few key environmental factors (Roling-Wilfred et

al 2002) that may be optimized to achieve a more efficient process The molar ratio of

carbon nitrogen and phosphorus (CNP) is very important for the metabolism of the

microorganisms and so for PAH degradation (Bossert amp Bartha 1984 Alexander 1994

Kwok amp Loh 2003) The molar ratio 100101 is frequently considered optimal for

contaminated soils (Bossert amp Bartha 1984 Alexander 1994) while other author have

reported negative or non-effects (Chaicircneau et al 2005) According to Leys et al (2005)

these contradictory results are due to the nutrients ratio required by PAH degrading bacteria

depends on environmental conditions type of bacteria and type of hydrocarbon In addition

the chemical form of those nutrients is also important being the soluble forms (ie iron or

nitrogen in form of phosphate nitrate and ammonium) the most frequent and efficient due to

their higher availability for microorganisms Depending on the microbial community and their

abundance another factor that may improve the PAH degradation is the addition of readily

assimilated such as glucose carbon sources (Zaidi amp Imam 1999)

Moreover the pH is an important factor that affects the solubility of both PAH and

many chemical species in the cultivation broth as well as the metabolism of the

microorganisms showing an optimal range for bacterial degradation between 55 and 78

(Bossert amp Bartha 1984 Wong et al 2001)

In general bioremediation process optimization may be flawed by the lack of studies

showing the simultaneous effect of different environmental factors Hence our main goal was

to set up the optimum values of six abiotic factors CNP molar ratio nitrogen source iron

source iron concentration pH and carbon source for the biodegradation of three PAH

Capiacutetulo 1a Optimisation of abiotic factors of PAH biodegradationprocess

52

(naphthalene phenanthrene and anthracene) at 25 ordmC In order to achieve the main objective

we analyzed the effects of the above factors on the microbial growth and the biotic

degradation rate

Materials and methods

Chemicals and media

Naphthalene phenanthrene and anthracene (all gt99 purity) were purchased from Sigma-

Aldrich (Steinheim Germany) and Fluka (Steinheim Germany) The consortium C2PL05

was not able to degrade PAH significantly without the addition of surfactants (data not

shown) Therefore surfactant Tween-80 (Sigma-Aldrich Steinheim Germany) was selected

as the most efficient biodegradable and non-toxic surfactant (Bautista et al 2009) for the

consortium C2PL05 Bushnell Haas Broth medium (BHB) was purchased from Panreac

(Barcelona Spain) and its composition is 02 gmiddotl-1 MgSO4middot7H2O 002 gmiddotl-1 CaCl2middot2H2O 1 gmiddotl-

1 KHPO4 1 gmiddotl-1 K2HPO4 1 gmiddotl-1 NH4NO3 005 gmiddotl-1 FeCl3 This base composition was

modified in each experiment as required

PAH degrader consortium C2PL05

The consortium C2PL05 was obtained from a soil sample in a petrochemical complex in

Puertollano Spain To obtain the consortium 1g of soil (lt 2 mm) was resuspended in 10 ml

of phosphate buffer saline (PBS) and incubated during 12 h in an orbital shaker (Innova 40

New Brunswick Scientific Edison NJ USA) at 150 rpm and 25ordmC under dark conditions

After that 15 ml of the supernatant was inoculated in 50 ml of BHB broth (pH 70) 1 wt

Tween-80 as surfactant and naphthalene phenanthrene and anthracene (each at 500 mgmiddotl-1)

as carbon source The culture was incubated at 150 rpm and 25ordmC under dark conditions

until the exponential phase was completed This was confirmed by monitoring the cell density

by absorbance at 600 nm in a spectrophotometer (Spectronic GenesysTM UK) Then the

consortium was stored at 4 ordmC to stop its growth At the beginning of each experiment 500 μl

of the stored consortium was inoculated into the fermentation flasks To identify the microbial

consortium C2PL05 colonies from aliquots of the soil extract were isolated in BHB agar

plates with PAH as only carbon source to confirm that these colonies were PAH degraders

Eight colonies were isolated and transferred onto LB-glucose agar plates in order to increase

microbial biomass for DNA extraction Total DNA of the colonies was extracted using

Microbial DNA isolation kit (MoBio Laboratories) Amplification of the 16S rRNA coding


53

region of the DNA was performed as described by Vintildeas et al (2005) using the primers

16F27 and 16R1488 Sequences were edited and assembled using BioEdit 487 software

(Hall 1999) All isolated strains of the consortium C2PL05 were γ-proteobacteria and the

genera present were Enterobacter Pseudomonas and Stenotrophomonas In addition non

culture-dependent molecular techniques as denaturant gradient gel electrophoresis (DGGE)

was performed to know the total biodiversity of the microbial consortium C2PL05 16S rRNA

gen was amplified using the primers 341F-GC and 907R (GC clamp 5acute-CGC CCG CCG

CGC CCC GCG CCC GTC CCG CCC CCG CCC-3acute) (Muyzer et al 1995) About 6 of

polyacrylamide (3751 acrylamidebisacrylamide) gels with a 30-60 urea-formamide

denaturant gradient and 075 mm were used in 1xTAE buffer at 200V for 4h at 60 ordmC The

bands were excised and reamplificated to identify the DNA The two genera identified

coincided with genera Pseudomonas and Stenotrophomonas identified by culture-dependent

techniques (more details in Molina et al 2009)

Experimental design

A total of 6 abiotic factors were evaluated To obtain an optimum value three treatments

each in triplicate were performed for each factor The replicates were carried out in 100 ml

Erlenmeyer flasks with 50 ml of BHB medium (pH 70) Tween-80 (1wt) naphthalene

phenanthrene and anthracene (each at 500 mgmiddotl-1) and 500 microl of the C2PL05 consortium

The concentration of the inoculum was 315x106 cells ml-1 of the heterotrophic microorganism

and 695x105 cells ml-1 of the PAH degrading microorganism The number of the

microorganisms capable to degrade any carbon source present in the medium (heterotrophic

microorganisms) and microorganisms capable to degrade PAH as sole carbon source (PAH-

degrading bacteria) were measured by the most probably number (MPN) method (Wrenn amp

Venosa 1996) LB-glucose broth and BHB medium were used to determine heterotrophic

microorganism and PAH degrading microorganism respectively To maintain the same initial

number of cells in each experiment the absorbance of the inoculum was measured and

diluted if necessary before inoculation to reach an optical density of 16 AU The replicates

were incubated in an orbital shaker (Innova 40 New Brunswick Scientific Edison NJ USA)

at 150 rpm and 25 ordmC under dark conditions Previous to inoculate the consortium the

Erlenmeyer flasks were shacked overnight to solubilize most of the PAH Samples were

withdrawn at 0 15 24 39 48 64 72 159 and 168 h to monitor the PAH depletion and cell

growth


54

Treatment conditions

Composition of BHB base was 02 gmiddotl-1 MgSO4middot7H2O 002 gmiddotl-1 CaCl2middot2H2O 1 gmiddotl-1 KHPO4 1

gmiddotl-1 K2HPO4 1 gmiddotl-1 NH4NO3 005 gmiddotl-1 FeCl3 The compounds MgSO4 and CaCl2 and their

concentration were constant for all treatments and KHPO4 and K2HPO4 were modified only in

concentration The other components were modified both the concentration and compounds

according to the requirements of the optimized factors PAH at 1500 mgmiddotl-1 (500 mgmiddotl-1 of

naphthalene phenathrene and anthracene) was used as carbon source for all treatments

except for those in which the carbon source was optimized and PAH were mixed with

glucose in a proportion of 50 PAH-glucose or only glucose was added In all cases an

overall carbon concentration of 01176 mmoll-1 was used Once a factor was optimized its

optimum value was kept for the subsequent factor optimization

The levels of each factor studied were selected as described below For the CNP

molar ratio the values employed were 100101 frequently described as optimal (Bossert

and Bartha 1984) 100505 and 1002116 To optimize the nitrogen source NaNO3

NH4(NO3) and (NH4)2SO3 were used The optimal iron source was selected amongst FeCl3

Fe(NO3)3 and Fe2(SO4)3 The concentration levels of the optimal iron form were 005 01 and

02 mmoll-1 and three different pH values were also tested 50 70 and 80 The effect of the

carbon source was determined by adding PAH as only carbon source PAH and glucose

(50 of carbon atoms from each source) or glucose as only carbon source

Bacterial growth

Bacterial growth during the PAH degradation process was monitored at 0 15 24 39 48 64

72 159 and 168 h by spectrophotometric absorbance of the culture media at 600 nm in a

UV-Vis spectrophotometer (Spectronic GenesysTM UK) From the above optical density data

the average of the cell density increments (CDI) was calculated by applying the following

equation

1

1

ii

iii tt

AlnAlnexpA Equation 1

where A is the absorbance at 600 nm t is the time elapsed in hours and the subscript i

corresponds to each sample or sampling time


55

Kinetic degradation

Naphthalene phenanthrene and anthracene concentrations in the culture media were

analysed using a ProStar 230 HPLC system (Varian Palo Alto CA USA) with a reverse

phase C18 column following the method described in Bautista et al (2009) The

concentration of each PAH was calculated from a standard curve based on peak area using

the absorbance at 254 nm Depletion rate of each PAH (-ri) during the experiments was fitted

to a first order kinetic model (Equation 2)

iBiiAii

i CkCkdt

dCr Eq 2

where C is the concentration of the corresponding PAH kA is the apparent first-order

kinetic constant due to abiotic processes kB is the apparent first-order kinetic constant

due to biological processes t is the time elapsed and the subscript i corresponds to

each PAH

Degradation caused by abiotic processes was determined by control experiments

carried out in triplicate in 100 ml Erlenmeyers flask with 50 ml of BHB medium (pH 70)

Tween-80 (1 wt) naphthalene phenanthrene and anthracene (each at 500 mgmiddotl-1) without

any microbial inoculum in an orbital shaker (Innova 40 New Brunswick Scientific Edison

NJ USA) at 150 rpm and 25 ordmC under dark conditions PAH concentration in the control

experiment were analysed using the HPLC system described previously The values of kA for

each PAH was calculated by applying eq 2 considering kB asymp 0 since no bacterial consortium

was inoculated

Statistical analysis

In order to evaluate the effects of the treatments on the kinetic biodegradation constant (kB)

and cell density increments (CDI) bifactorials analysis of variance (ANOVA) were used The

variances were checked for homogeneity by applying the Cochranacutes test When indicated

data were transformed to homogenize variances Student-Newman-Keuls (SNK) test was

used to discriminate among different treatments after significant F-test All tests were

performed with the software Statistica 60 for Windows


56

Results

Control experiments (Figure 1) show that phenathrene and anthracene concentration was

not affected by any abiotic process since no depletion was observed along the experiment

so that kA asymp 0 h-1 However in the case of naphthalene some degree of abiotic depletion was

measured during the controls yielding an apparent first-order abiotic rate constant of 27x10-

3 plusmn 7x10-5 h1 This value was accounted for the calculation of the biodegradation rate

constant (kB) for naphthalene in the optimisation experiments

0 100 200 300 400 500 600 700

20

40

60

80

100

Rem

aini

ng P

AH

(

)

Time (hour)

Figure 1 Naphthalene ( ) phenathrene ( ) and anthracene ( )

depletion due to abiotic processes in control experiments

Table 1 Analysis of variance (ANOVA) summary for the cell density increments (CDI) and the

biotic degradation constant (kB) MS is the means of squares and df degrees of freedom

CDI kB

Factor df MS F-value p-value df MS F-value p-value

CNP ratio 2 27middot10-1 238 4 57middot10-2 566 Error 6 11middot10-2 18 10middot10-3

N source 2 21middot10-1 234 4 90middot10-6 113

Error 6 10middot10-2 18 70middot10-7

Fe source 2 18middot10-2 51 4 30middot10-6 43


Fe Concentration 2 45middot10-1 348 006 4 30middot10-6 38


pH 2 30middot10-2 1103 4 15middot10-4 5


GlucosePAHs 2 54middot10-1 45401 2 66middot10-4 7


a Logarithmically transformed data to achieve homogeneity of variance


57

Cell density increments of the consortium for three different treatments of CNP molar

ratio are showed in Figure 2A According to statistical analysis of CDI there was significant

differences between CNP molar ratio (F26 = 238 p lt 001 Table 1) and SNK showed that

treatments with molar ratios of 100101 and 1002116 reached larger increases With

regard to the kinetic biodegradation constant (kB) the interaction between kB of the

treatments with each hydrocarbon was significant (F418 = 57 p lt 0001 Table 1) The SNK

test (Figure 2B) showed that the treatment 1002116 with naphthalene yielded the highest

value whereas the lowest were achieved with 100505 and 100101 for anthracene and

phenanthrene In addition within each PAH group the highest values were observed with

1002116 molar ratio Therefore although there are no differences for CDI between ratios

100101 and 1002116 1002116 molar ratio is the most effective for the PAH degradation

so that this ratio was considered as the optimal

171819202122232425

100101 1002116100505

bb

a

A

CNP molar ratio

CD

I

Naphthalene Phenanthrene Anthracene-35

-30

-25

-20

-15

-10

-05

00B

d

g

e

bc

f

ab

f

Log

k B (

h-1)

Figure 2 (A) Cell density increments of the consortium C2PL05 with the treatments 100505

100101 and 1002116 Error bars show the standard error (B) Differences between treatments

(100101 100505 and 1002116 ) and PAHs in the biodegradation kinetic constant (kB)

The letters show differences between groups (p lt 005 SNK) and the error bars the standard

deviation


58

Figure 3A shows that the three different nitrogen sources added had significant effects

on CDI (F26 = 234 p lt 001 Table 1) The SNK test shows that the addition of NaNO3

significantly improved CDI The interaction between PAH and the nitrogen sources were

significant (F418 = 113 p lt 0001 Table 1) and the highest kB values were achieved with

NaNO3 for naphthalene phenanthrene and anthracene (Figure 3B) According to these

results NaNO3 is considered as the best form to supply the nitrogen source for both PAH

degradation and growth of the C2PL05 consortium

19

20

21

22

23

24

25

(NH4)

2SO

4NH4NO

3NaNO

3

a

b

a

A

Nitrogen source

CD

I

Naphthalene Phenanthrene Anthracene0

2x10-3

4x10-3

6x10-3

8x10-3

1x10-2

Bf

ba

e

bcb

dbc

a

kB (

h-1)

Figure 3 (A) Cell density increments of the consortium C2PL05 with the treatments NaNO3 NH4NO3

and (NH4)2SO4 Error bars show the standard error (B) Differences between treatments (NaNO3

NH4NO3 and (NH4)2SO4 ) and PAHs in the biodegradation kinetic constant (kB) The letters show

differences between groups (p lt 005 SNK) and the error bars the standard deviation


59

CDI of the treatments performed with three different iron sources (Figure 4A) were

significantly different (F26 = 51 p lt 005 Table 1) Although no significant differences

between adding Fe2(SO4)3 or Fe(NO3)3 were observed the addition of Fe2(SO4)3 contributes

more to CDI than FeCl3 The kB (Figure 4B) showed significant differences in the interaction

between PAH and the different iron sources (F418 = 43 p lt 0001 Table 1) The highest kB

values were observed with Fe2(SO4)3 for the degradation of phenanthrene followed by FeCl3

degrading naphthalene and phenanthrene The lowest values of kB were observed with

Fe(NO3)3 degrading naphthalene and anthracene Nevertheless the most recalcitrant PAH

(phenanthrene and anthracene) showed the highest kB values with Fe2(SO4)3 in agreement

with the highest CDI values also obtained with Fe2(SO4)3

168

172

176

180

184

188

192

196

Fe(NO3)

3 Fe2(SO

4)

3FeCl

3

ab

b

a

A

Iron source

CD

I


2x10-3

4x10-3

6x10-3

8x10-3

1x10-2

B

c

a

b

c

b

d

b

a a

k B

(h-1

)

Figure 4 (A) Cell density increments of the consortium C2PL05 with the treatments FeCl3 Fe(NO3)3

and Fe2(SO4)3 Error bars show the standard error (B) Differences between treatments (FeCl3

Fe(NO3)3 and Fe2(SO4) ) and PAHs in the biodegradation kinetic constant (kB) The letters show



60

Concerning the effect of the iron concentration (Figure 5) supplied in the form of the

optimal Fe2(SO4)3 no significant differences in CDI were found for all three concentration

used (F26 = 348 p = 006 Table 1 Figure 5A) However the interaction between iron

concentration and kB of three PAH was significant (F418 = 38 p lt 0001 Table 1) reaching

the highest values for kB by using an iron concentration of 01 mmoll-1 degrading

naphthalene and phenanthrene (Figure 5B) The lowest values of kB were observed with 005

mmoll-1 and 02 mmoll-1 degrading phenanthrene and anthracene (Figure 4B) Since each

PAH showed the highest kB with 01 mmoll-1 this iron concentration was considered as the

most efficient for the PAH biodegradation process

005 01 02

38

40

42

44

46

48

50

a

a

a

A

Iron concentration (mmol l-1)

CD

I


50x10-3

10x10-2

15x10-2

20x10-2

B

c

f

d

b

e

d

cb

a

k B (

h-1)

Figure 5 (A) Cell density increments of the consortium C2PL05 with the treatments 005 mmolmiddotl-1 01

mmolmiddotl-1 and 02 mmolmiddotl-1 Error bars show the standard error (B) Differences between treatments

(005 mmolmiddotl-1 01 mmolmiddotl-1 and 02 mmolmiddotl-1 ) and PAHs in the biodegradation kinetic

constant (kB) The letters show differences between groups (p lt 005 SNK) and the error bars the

standard deviation


61

With reference to pH Figure 6A and statistical analysis (F26 = 1103 p lt 001 Table 1)

clearly show that the neutral pH of the medium favour the CDI of the consortium The kB of

the three different treatments (Figure 6B) also showed significant differences in the

interaction (F49 = 5 p lt 005 Table 1) The highest value of kB was observed for anthracene

degradation at neutral pH (Figure 6B) The other two PAH naphthalene and phenanthrene

did not show significantly differences between any treatments Therefore given that the

highest values of both parameters (CDI and kB) were observed at pH 7 this value will be

considered as the most efficient for the PAH biodegradation process

5 7 8

215

220

225

230

235

240

245

a

b

a

A

pH

CD

I


50x10-3

10x10-2

15x10-2

20x10-2

25x10-2

30x10-2

B

b

a

ab ab

a

ab

c

ab ab

kB

(h-1

)

Figure 6 (A) Cell density increments of the consortium C2PL05 with the treatments pH 50 pH 70

and pH 80 Error bars show the standard error (B) Differences between treatments (pH 50 pH

70 and pH 80 ) and PAHs in the biodegradation kinetic constant (kB) The letters show



62

The last factor analyzed was the addition of an easily assimilated carbon source

(Figure 7) Regarding to CDI values (Figure 7A) there were significant differences between

treatments (F26 = 45401 p lt 0001 Table 1) The addition of glucose as only carbon source

significantly improved CDI Figure 7B only show the kB of the treatments with PAH (100 or

50 of PAH) therefore the treatment with glucose as only carbon source was not included in

the ANOVA analysis The interaction between PAH and type of carbon source was

significant (F212 = 7 p lt 005 Table 1) The kB for the treatment with PAH and glucose

(5050) was significantly higher for phenanthrene and naphthalene (Figure 6B) although

there were no differences with the treatment for anthracene where PAH were the only carbon

source

PAHs (100)

PAHsGlucose (50)Glucose (100)

18

20

22

24

26

28

Carbon source

b

c

a

A

CD

I


2x10-2

4x10-2

6x10-2

8x10-2

1x10-1

B

c

bb

b

b

a

k B (h

-1)

Figure 7 (A) Cell density increments of the consortium C2PL05 with the treatments PAHs (100)

PAHsglucose (5050) and glucose (100) Error bars show the standard error (B) Differences

between treatments (PAHs (100) and PAHs glucose (5050) ) and PAHs in the

biodegradation kinetic constant (kB) The letters show differences between groups (p lt 005 SNK)

and the error bars the standard deviation


63

Discussion

It is important to highlight that the increments of the cell density is a parameter that brings

together all the microbial community whereas the biotic degradation constant is specific for

the PAH degrading microorganisms For that reason when the effect of the factors studied

on CDI and kB yielded opposite results the latter always prevailed since PAH degradation

efficiency is the main goal of the present optimisation study

With regard to the CNP molar ratio some authors consider that low ratios might limit

the bacterial growth (Leys et al 2005) although others show that high molar ratios such as

100101 are optimum for hydrocarbon polluted soils (Flathman et al 1994 Bouchez et al

1995 Eweis et al 1998) However in agreement with Leys et al (2005) our results

confirmed that the most effective molar ratio was the highest (1002116) This result

suggests that the supply of the inorganic nutrients during the PAH biodegradation process

may be needed by the microbial metabolism In addition the form used to supply these

nutrients can affect the metabolism of the microorganism (Carmichael amp Pfaender 1997) and

limit the amount of carbon that bacteria can assimilate limiting in turn the biodegradation

extent Our results showed that nitrate (sodium nitrate) as nitrogen source improved PAH

biodegradation as compared to ammonium This is likely due to the fact that nitrate is more

soluble and available for microorganisms than ammonium which has adsorbent properties

(Schlessinger 1991) The iron is other essential compound to stimulate the microbial activity

on PAH degradation (Dinkla amp Janssen 2003 Santos et al 2008)

On one hand iron acts as a cofactor of enzymes catalysing PAH oxidation (Dinkla amp

Janssen 2003) but it is also related with the production of biosurfactants (Santos et al

2008) These compounds are naturally produced by genera such as Pseudomonas and

Bacillus (Wei et al 2003) increasing the PAH solubility and therefore their bioavailability In

agreement with previous works (Dinkla amp Janssen 2003 Santos et al 2008) our results

confirmed that the addition of iron in a concentration of 01 mmoll-1 makes the

biodegradation more effective Santos et al (2008) stated that there is a limit concentration

above which the growth is inhibited due to toxic effects According to these authors our

results showed lower degradation and growth with the concentration 02 mmoll-1 since this

concentration may be saturating for these microorganisms However opposite to previous

works (Dinkla amp Janssen 2003 Santos et al 2008) the most effective iron form was

Fe2(SO4)3 for the PAH biodegradation likely due to the higher solubility which makes it more

available for the microorganism


64

The addition of easy assimilated carbon forms such as glucose for the PAH

degrading process can result in an increment in the total number of bacteria (Wong et al

2001) because PAH degrader population can use multiple carbon sources simultaneously

(Herwijnen et al 2006) However this increment in the microbial biomass was previously

considered (Wong et al 2001) because the utilization of the new carbon source may

increase the lag phase delaying the bacterial growth (Maier et al 2000) Our results

confirmed that PAH degradation was more efficient with the addition of an easy assimilated

carbon source probably because the augmentation of the total heterotrophic population also

enhanced the PAH degrading community Our consortium showed a longer lag phase during

the treatment with glucose than that observed during the treatment with PAH as only carbon

source (data not shown) These results are consistent with a consortium completely adapted

to PAH biodegradation and its enzymatic system requires some adaptation time to start

assimilating the new carbon source (Maier et al 2000)

Depending on the type of soil and the type of PAH to degrade the optimum pH range

can be very variable (Dibble amp Bartha 1979) Some acid resistant gram-positive bacteria

such as Mycobacterium sp show better PAH degradation capabilities under acid condition

because and low pH seems to render the mycobacterial more permeable to hydrophobic

substrates (Kim et al 2005) However other microorganisms belonging to Pseudomonas

genus prefer neutral pH conditions In agreement with previous works (ie Dibble amp Bartha

1979) our results confirmed that neutral pH is optimum for the biodegradation PAH

In summary the current work has shown that the optimization of environmental

parameters may significantly improve the PAH biodegradation process It is also important to

underline that the statistical analysis of data and the combined study of the bacterial growth

and the kinetics of the degradation process provide an accurate interpretation of the

optimisation results Concluding for an optimum bioremediation process is very important to

perform these previous bioassays to decrease the process development time and so the

associated costs

Acknowledgements

This work has been funded by the Spanish Ministery of Enviroment (11-37320053-B and

0120062-11) and by Fundacioacuten Alfonso Martiacuten Escudero The consortium C2PL05 was

isolated from soil samples kindly provided by Repsol SA This work is framed within the

Official Master en Ciencia y Tecnologiacutea Ambiental of the Universidad Rey Juan Carlos


65

References

Alexander M 1994 Biodegradation and Biorremediation Academic Press New York

Bautista LF Sanz R Molina MC Gonzaacutelez N amp Saacutenchez D 2009 Effect of different non-

ionic surfactants on the biodegradation of PAH by diverse bacteria Int Biodeter

Biodegr 63 913-922

Bossert I amp Bartha R 1984 The fate of petroleum in soil ecosystems In Atlas RM (ed)

Petroleum microbiology Macmillan New York pp441-4473

Bouchez M Blanchet D amp Vandecasteele J-P 1995 Degradation of polycyclic aromatic

hydrocarbons by pure strains and by defined strain associations inhibition

phenomena and cometabolism Appl Environ Microbiol 43 156-164

Carmichael LM amp Pfaender KF 1997 The effects of inorganic and organic supplements on

the microbial degradation of phenanthrene and pyrene in soils Biodegradation 8 1-

13

Dibble JR amp Bartha R 1979 Effect of environmental parameters on the biodegradation of

oil sludge Appl Environ Microbiol 37 729-739

Dinkla EJT amp Janssen DB 2003 Simultaneous growth on citrate reduces the effects of

iron limitation during toluene degradation in Pseudomonas Microb Ecol 45 97-107

Eweis JB Ergas SJ Chang PY amp Schroeder ED 1998 Bioremediation Principles

McGraw-Hill Boston pp 136-236

Flathman PE Jerger DE amp Exner JH1994 Biorremediation-Field Experiences Lewis

Publishers Boca Raton pp 81-106 383-490

Gallego JL Garciacutea MJ Llamas JF Belloch C Pelaez AI amp Sanchez J 2007

Biodegradation of oil tank botton sludge using microbial consortia Biodegradation 18

269-281

Hall TA 1999 Bioedit a user-friendly biological sequence alignment editor and analysis

program for Windows 9598NT Nucleic Acids Symp Ser 41 95-98

Heitkamp MA amp Cerniglia CE 1988 Mineralization of polycyclic aromatic hydrocarbons by

a bacterium isolated from sediment below an Oil Field Appl Environ Microbiol 54

1612-1614

Kim YH Freeman JP Moody JD Engesse KH amp Cerniglia CE 2005 Effects of pH on

the degradation of phenanthrene and pyrene by Mycobacterium vanbaalenii PYR-1

Appl Environ Microbiol 67 275-285

Kwok Chen-Ko amp Loh Kai-Che 2003 Effects of Singapore soil type on bioavalability of

nutrients in soil bioremediation Adv Environ Res 7 889-900


66

Leys MN Bastiaens L Verstraete W amp Springael D 2005 Influence of the

carbonnitrogenphosphorus ratio on polycyclic aromatic hydrocarbons degradation

by Mycobacterium and Sphingomonas in soil Appl Microbiol Biot 66 726-736

Luning Prak DJ amp Pritchard PH 2002 Solubilization of polycyclic aromatic hydrocarbon

mixtures in micelar non-ionic surfactant solution Water Res 36 3463-3472

Maier MR Pepper LI amp Gerba PC 2000 Enviromental Microbiology Academic Press

Elsevier

Molina MC Gonzalez N Bautista LF Sanz R Simarro R Saacutenchez I amp Sanz L 2009

Isolation and genetic identification of PAH degrading bacteria from a microbial

consortium Biodegradation 20 789-800

Muyzer G Hottentrager S Teske A amp Wawer C 1995 Denaturing gradient gel

electrophoresis of PCR-amplified 16S Rdna a new molecular approach to analyse the

genetic diversity of mixed microbial communities In Akkermans ADL van Elsas JD

de Bruijn FJ (eds) Molecular microbial ecology manual Kluwer Academic Publishers

Dordrecht pp 1-23

Rolling-Wilfred FM Milner M Jones DM Lee K Daniel F Swanell-Richard JP amp Head

IM 2002 Robust hydrocarbon degradation and dynamic of bacterial communities

during nutrients-enhanced oil spillbiorremediation Appl Environ Microbiol 68 5537-

5548

Santos EC Jacques JS Bento MF Peralba MCR Selbach AP Saacute LS amp Camargo

AOF 2008 Anthracene biodegradation an surface activity by an iron-stimulated

Pseudomonas sp Bioresource Technol 99 2644-2649

Schlessinger WH 1991 Biogeochemistry Academic Press San Diego

Vintildeas M Sabateacute J Guasp C Lalucat J y Solanas AM 2005 Culture-dependent and

independent aproaches establish the complexity of a PAH degrading microbial

consortium Can J Microbiol 51 897-909

Wei Y-H Wang LF Chang JS amp Kung SS 2003 Identification of induced in iron enriched

cultures of Bacillus subtilis during biosurfactant fermentation J Biosci Bioeng 96

174-178

Wong JWC Lai KM Wan CK Ma KK amp Fang M 2001 Isolation and optimization of

PAH-degradative bacteria from contaminated soil for PAH bioremediation Water Air

Soil Poll 13 1-13

Zaidi BR amp ImamSH 1999 Factors affecting microbial degradation of polycyclic aromatic

hydrocarbon phenanthrene in caribbean coastal water Mar Pollut Bull 38 738-749

Capiacutetulo

Aceptado en Water Air amp Soil Pollution (Febrero 2012)

Simarro R Gonzaacutelez N Bautista LF Molina MC amp Schiavi E

Evaluation of the influence of multiple environmental factors on the biodegradation

of dibenzofuran phenanthrene and pyrene by a bacterial consortium using an orthogonal

experimental design

Evaluacioacuten de la influencia de muacuteltiples factores ambientales en la biodegradacioacuten de dibenzofurano

fenantreno y pireno por un consorcio bacteriano usando un disentildeo experimental ortogonal

1b

Capiacutetulo 1b Influence of abiotic factors on the PAH biodegradation using an orthogonal design

69

Abstract

For a bioremediation process to be effective we suggest to perform preliminary studies in

laboratory to describe and characterize physicochemical and biological parameters (type and

concentration of nutrients type and number of microorganisms temperature) of the

environment concerned We consider that these studies should be done by taking into

account the simultaneous interaction between different factors By knowing the response

capacity to pollutants it is possible to select and modify the right experimental conditions to

enhance bioremediation


71

Introduction

Polycyclic aromatic hydrocarbons (PAH) are a group of organic compounds composed of two

or more aromatics rings High molecular weight PAH (HMW-PAH) with four (ie pyrene) or

more aromatics rings and other heterocyclic aromatic compounds as dibenzofuran both with

high molecular mass are often more difficult to biodegrade that other low molecular weight

PAH (LMW-PAH) due to their lower solubility and biodegradability Many of them have toxic

mutagenic and carcinogenic properties and the effects of PAH as naphthalene or

phenanthrene in animals and humans their toxicity and carcinogenic activity has been

reported and well documented (Sudip et al 2002) In addition PAH are bioaccumulated in

the environment and trophic chains properties that increase with the numbers of rings There

is a natural degradation carried out by microorganism able to use PAH as carbon source

which represents a considerable portion of the bacterial communities present in polluted soils

(Heitkamp amp Cerniglia 1998) However this natural biodegradation may be affected by

environmental factors which optimization allows us to achieve a more efficient process

Temperature is a key factor in the physicochemical properties of PAH as well as in the

metabolism of the microorganisms Although it has been shown that biodegradation of PAH

is possible even at temperatures lower than 5 ordmC (Eriksson et al 2001) it is usually more

efficient at mild temperatures (15-25 ordmC) (Mohn amp Stewart 2000) The carbon nitrogen and

phosphorus (CNP) molar ratio is another important factor in biodegradation process

because affect the dynamics of the bacterial metabolisms changing the PAH conversion

rates and growth of PAH-degrading species (Leys et al 2004) The form in which these

essential nutrients are supplied affects the bioavailability for the microorganism being more

soluble and efficient the oxidated forms (such as nitrates) than reduced forms (such as

ammonium) (Schlessinger 1991)

Surfactants are compounds used to increase the PAH solubility although both

positive (Boochan et al 1998 Jin et al 2007) and negative effects (Boochan et al 1998

Laha amp Luthy 1992) on the biodegradation process has been reported The nature of the

effect depends on several factors such as the type and concentration of surfactant due to

the toxic properties of some of them (Jin et al 2007) and the increasing of toxicity of PAH

produced by increasing their solubility (Thibault et al 1996) Another factor considered is the

inoculum size related to the diversity and effectiveness of the biodegradation because in a

diluted inoculum the minority microorganisms which likely have an important role in the

biodegradation process can be removed (Szaboacute et al 2007) Moreover it has been

reported (Szaboacute et al 2007) that the addition of a readily metabolized carbon source (ie

glucose) improves the PAH degradation possibly due to the increased biomass although in

72

others cases (Wong et al 2000) this better bacterial growth reduced significantly PAH

degradation

We consider that the study of the individual effect of abiotic factors on the

biodegradation capacity of the microbial consortium is incomplete because the effect of one

factor can be influenced by other factors In this work the combination between factors was

optimized by an orthogonal experimental design fraction of the full factorial combination of

the selected environmental factors

Hence our two mains goals are to determine the optimal conditions for the

biodegradation of low (phenanthrene and dibenzophurane) and high (pyrene) molecular

weight PAH by a bacterial degrading consortium (C2PL05) and the study of the influence of

the factors (temperature CNP molar ratio type of nitrogen and iron source iron source

concentration carbon source surfactant concentration and inoculums dilution) in the

biodegradation In order to achieve these objectives we realized an orthogonal experimental

design to take into account all combination between eight factors temperature CNP molar

ratio nitrogen and iron source iron concentration addition of glucose surfactant

concentration and inoculum dilution at three and two levels

Material and methods

Chemicals and media

Dibenzofuran phenanthrene and pyrene (gt99 purity) were purchased from Sigma-Aldrich

Steinheim Germany) Stock mix of the three PAH was prepared by dissolving the necessary

amount in n-hexane (Fluka Steinheim Germany) In previously work (Bautista et al 2009)

we tested that the optimal surfactant for the consortium was the biodegradable and non

toxicTween-80 (Sigma-Aldrich Steinheim Germany) Bushnell-Haas Broth medium (BHB)

was purchased from Panreac (Barcelona Spain) and its original composition (02 g l-1

MgSO4middot7H2O 002 g l-1 CaCl2 2H2O 1 g l-1 KHPO4 1 g l-1 K2HPO4 1 g l-1 NH4NO3 005 g l-1

FeCl3) was modified according to the treatment (see Table 1)


73

Table 1 Experimental design

Treatment T

(ordmC) CNP (molar)

N source

Fe

source

Iron source concentration

(mM)

Glucose PAH ()

Surfactant concentration

Inoculum dilution

1 30 100505 (NH4)2SO3 Fe2(SO4)3 02 0100 CMC 10-3

2 20 1002116 (NH4)2SO3 FeNO3 005 0100 + 20CMC 10-2

3 25 100101 NaNO3 FeNO3 02 0100 + 20CMC 10-1

4 20 100505 NaNO3 Fe2(SO4)3 02 5050 + 20CMC 10-2

5 25 100505 NH4NO3 FeNO3 01 5050 CMC 10-2

6 30 100101 NH4NO3 Fe2(SO4)3 005 8020 + 20CMC 10-2

7 30 100101 NaNO3 FeCl3 01 0100 CMC 10-2

8 20 100505 NaNO3 FeCl3 005 8020 CMC 10-1

9 25 1002116 (NH4)2SO3 FeCl3 02 8020 CMC 10-2

10 20 1002116 NH4NO3 Fe2(SO4)3 01 0100 CMC 10-1

11 20 100101 NH4NO3 FeNO3 02 8020 CMC 10-3

12 25 100101 (NH4)2SO3 Fe2(SO4)3 005 5050 CMC 10-1

13 25 1002116 NaNO3 Fe2(SO4)3 01 8020 + 20CMC 10-3

14 30 1002116 NH4NO3 FeCl3 02 5050 + 20CMC 10-1

15 25 100505 NH4NO3 FeCl3 005 0100 + 20CMC 10-3

16 30 1002116 NaNO3 FeNO3 005 5050 CMC 10-3

17 30 100505 (NH4)2SO3 FeNO3 01 8020 + 20CMC 10-1

18 20 100101 (NH4)2SO3 FeCl3 01 5050 + 20CMC 10-3

Bacterial consortium

PAH-degrading consortium C2PL05 was isolated from a soil in a petrochemical complex in

Puertollano (Spain) and was identified and described in Molina et al (2009) All strains of

the consortium C2PL05 isolated by culture-dependent techniques were γ-Proteobacteria

and the strains presents belong to the genera Enterobacter Pseudomonas and

Stenotrophomonas (Molina et al 2009) In addition the diversity of the enriched microbial

consortium was characterised by a non culture-dependent molecular technique such as

denaturing gradient gel electrophoresis (DGGE) following the procedure described

elsewhere (Molina et al 2009) using the primers 341F-GC and 907R (GC clamp 5acute-CGC

CCG CCG CGC CCC GCG CCC GTC CCG CCC CCG CCC-3acute) (Muyzer et al 1995)

Experimental design

An orthogonal design form of L18 (37) (21) selected from the module of Statistica (Version 60)

was used to do the multi-factor combination A total of 18 experiments each in triplicate

were carried out in 100 ml Erlenmeyers flask with a total volume of 50 ml of Bushnell-Haas

Broth medium (BHB) (Panreac Barcelona Spain) with an original composition modified

74

according to the treatments requirements (see Table 1) The replicates were incubated in an

orbital shaker (Innova 40 New Brunswick Scientific Edison NJ USA) at 150 rpm under dark

conditions but prior to inoculate the consortium the flasks were shaken overnight to

equilibrate and solubilize most of the PAH In Table 1 shows a summary of environmental

conditions and incubation of each treatment Tween-80 concentration was 0012 mM the

critical micellar concentration (CMC) 100 of PAH was equivalent to 03 g l-1 (01 g l-1 of

each PAH) The initial cell concentration of the inoculum consortium was determined by the

most probably number (MPN) method (Wrenn amp Venosa 1983) The number of heterotrophic

microorganisms (315x106 cell ml-1) was measured with Luria Base broth (LB Panreac

Barcelona Spain) with glucose as carbon source and the PAH degrading microorganisms of

the consortium (695x105 cell ml-1) with BHB with PAH mix as carbon source

Cell density

Bacterial density during the PAH degrading process was monitored at 0 15 24 39 48 63

72 87 95 and 159 h by the increase in absorbance of the culture media at 600 nm in a

spectrophotometer (Spectronic GenesysTM England) Throughout the cell growth curve we

calculated the average of the cell densities increments (CDI) applying the equation 1

1

1

ii

iii tt


where A is the absorbance at 600 nm t is the time elapsed in hours and i

corresponds to each sample or sampling time The increments were normalized by

the initial absorbance measurements to correct the effect of the inoculum dilution

PAH extraction and analysis

At the end of each experiment (159 hours) PAH were extracted with dichloromethane and

the residue precipitated was dissolved in 1 ml of acetonitrile for high performance liquid

chromatography (HPLC) analysis using a ProStar 230 HPLC system (Varian Palo Alto CA

USA) with a reversed phase C18 column following the method previously described (Bautista

et al 2009) The residual concentration of each PAH was calculated from a standard curve

based on peak area at a wavelength of 254 nm The average percentage of phenanthrene

pyrene and dibenzofuran and average percentage of total PAH degradation (PD) for each

treatment are shown in Table 2


75

Statistical analyses

The effect of the individual parameters on the CDI and on the PD were analysed by a

parametric one-way analysis of variance (ANOVA) The variances were checked for

homogeneity by the Cochranacutes test Students-Newman-Keuls (SNK) test was used to

discriminate among different variables after significant F-test When data were not strictly

parametric Kruskal-Wallis test and Tukey-type multiple comparison test were used

The orthogonal design to determine the optimal conditions for PAH biodegradation is

an alternative to the full factorial test which is impractical when many factors are considered

simultaneously (Chen et al 2008) However the orthogonal test allows a much lower

combination of factors and levels to test the effect of interacting factors

Results and discussion

The consortium C2PL05 degrade phenanthrene pyrene and dibenfuran efficiently in 159 h

(Table 2) and also other PAH as naphthalene and anthracene (Molina et al 2009) The

study of the influence of each factor in the total PD (Figure 1) showed that only the carbon

source influenced in this parameter significantly (Table 3) Results concerning to carbon

source showed that PD were higher when PAH were added as only carbon source (100 of

PAH) The reason why the PD did not show statistical significance between treatments

except for the relative concentration of PAH-glucose may be due to significant changes

produced in PD at earlier times when PAH were still present in the cultivation media

However the carbon source incubation temperature and inoculum dilution were factors that

significantly influenced CDI (Table 3 Figure 2)

76

Table 2 Final percentage degradation of

phenanthrene (Phe) pyrene (pyr) and dibenzofuran

(Dib) and total percentage degradation (total PD) for

each treatment

percentage degradation Treatment Phe Pyr Dib Total PD

1 965 883 864 904 2 969 950 833 917 3 966 895 845 902 4 972 915 921 872 5 969 904 950 882 6 982 935 995 852 7 964 883 859 902 8 977 953 964 823 9 976 936 984 825 10 970 910 895 925 11 979 968 986 888 12 966 889 920 850 13 978 930 993 835 14 966 897 943 871 15 963 881 898 914 16 963 886 951 867 17 977 954 986 861 18 976 930 967 915

The conditions corresponding to listed treatments

are presented in Table 1

100

50

5

100

101

100

211

6

CNP

20

ordmC

25ordmC

30ordmC

82

84

86

88

90

92 T (ordmC)

aa

a

aa

aa

aa

a

Tot

al P

D (

)

NaN

O3

NH

4NO

3

(NH

4)2S

O3

N source

FeC

L3

FeN

O3

Fe2

(SO

4)3

a

a

0acute05 0acute1

0acute2

Fe source

a

a

a

0 -

100

50 -

50

80 -

20

C Fe (mM)

a

b

c

CM

C

+ 2

0 C

MC

Gluc-PAHs

aa

10^-

1

10^-

2

10^-

3DilutionCMC

aa

a

Figure 1 Graphical analysis of average values of total percentage degradation (PD) under

different treatments and levels of the factors () represent the average of the total PD of the

treatments of each level Letters (a b and c) show differences between groups


77

Table 3 Analysis of variance (ANOVA) summary for the increments of cell density (CDI) and the total

percentage degradation (PD) of each factor MS is the mean of squares and df degrees of freedom

ANOVA of CDI ANOVA of total PD


T (ordmC) Error

2 056 1889 2 22 183 ns

51 002 51 12

Molar ratio CNP Error

2 003 069 ns 2 22 183 ns

51 005 51 12

N source Error

2 001 007 ns 2 214 177 ns 51 005 51 121

Fe source Error

2 003 066 ns 2 89 071 ns

51 005 51 126

Fe concentration Error

2 007 146 ns 2 118 095 ns 51 005 51 124

Glucose-PAH Error

2 024 584 2 1802

3085 51 004 51 395

8

CMC Error

1 001 027 ns 1 89 071 ns

52 005 52 125

Inoculum Dilutionb Error

2 331 a 2 113 091 ns 54 6614 51 125

a H-value obtained of Kruskal-Wallis test used for non parametric data Chi-square = 28 Overall

median = 044

p-value lt 001

p-value lt 0001

100

50

5

100

100

1

100

211

6

CNP

20

ordmC

25ordmC

30ordmC

16

17

18

19

20

21

a

a

aa

a

aa

a

c

bCD

I

NaN

O3

NH

4NO

3

(NH

4)2S

O3

N source

FeC

L3

FeN

O3

Fe2

SO

4

Fe source

a

a

0acute05 0acute1

0acute2

C Fe (mM)

a

a

a

0-10

0

50-5

0

80-2

0

Gluc-PAH

a

b

c

CM

C

+ 2

0 C

MC

CMC

aa

10^-

1

10^-

2

10^-

3

00

05

10

15

20

25

30

35C

DI n

orm

aliz

ed

DilutionT (ordmC)

b

a

a

Figure 2 Graphical analysis of average values of cell density increments (CDI) and normalized cell

density increments (CDI normalized) of different treatments and levels of the factors () represent the

average of the CDI or CDI normalized of the treatments of each level Letters (a b and c) show

differences between groups

78

The temperature range considered in the present study might not affect the

biodegradation process since it is considered narrow by some authors (Wong et al 2000)

Nevertheless we observed significant differences in the process at different temperatures

showing an optimum at 25 ordmC for our microbial consortium growth (Figure 2) whereas when

consortium was incubated at 20 ordmC and 30 ordmC microorganisms remained in lag phase These

results were in agreement with the fact that respiration increases exponentially with

temperature (Q10 relationship) (Lloyd amp Taylor 1994) but increasing or decreasing

temperature beyond the optimal value will cause a reduction in microbial respiration We

suggest that moderate fluctuation of temperatures affect microbial growth rate but not

degradation rates because degrading population is able to degrade PAH efficiently in a

temperature range between 20-30 ordmC (Sartoros et al 2005)

The nutrient requirements for microorganisms increase during the biodegradation

process so a low CNP molar ratio can result in a reduced of the metabolic activity of the

degrader microorganisms and thus reduce their potential degrader (Leys et al 2004)

According to this author CNP ratios above 100101 provide enough nutrients to metabolize

the pollutants However our results showed that the CNP ratios supplied to the cultures

even the ratio 100505 did not affect the CDI and total PD This results indicate that the

consortium C2PL05 is able to degrade PAH even under low nutrients conditions due to its

high adaptation to the hard conditions of a chronically contaminated soil The results

concerning the addition of different nitrogen and iron sources did not show significant

difference in CDI and total PD Other works (Schelessinger 1991 Santos et al 2008) have

suggested that the addition of nitrogen in form of nitrates (Schelessinger 1991) and the iron

in form of sulphates or chlorides (Santos et al 2008) is more effective due to their high

solubility

The addition of readily biodegradable carbon source as glucose to a polluted

environment is considered an alternative to promote biodegradation The easy assimilation of

this compound result in an increase in total biomass (heterotrophic and PAH degrader

microorganisms) of the microbial population thereby increasing the degradation capacity of

the community Piruvate are a carbon source that promote the growth of certain degrading

strains as Pseudomonas putida (Lee et al 2003) whereas salicylate induces the synthesis

and activation of degradative enzymes (Chen amp Aitken 1999) Similarly to previous results

observed by Wong et al (2000) in the present study the addition of glucose to the cultures

had significant effects in total PD and CDI (Figure 1 Figure 2) Although the consortium

C2PL05 showed a significantly better growth with 80 of glucose the difference between

treatments (0100 5050 8020 of glucosePAH) showed that PD was higher when PAH

were added as only carbon source Previously it has been described that after a change in


79

the type of carbon source supplied to PAH-degrader microorganisms an adaptation period

for the enzymatic system was required reducing the mineralization rate of pollutants (Wong

et al 2000 Maier 2009 Simarro et al 2010) As glucose was added as additional carbon

source our results show an increase in CDI although the PD values decrease significantly

This indicated that glucose enhance the overall growth of consortium but decrease the

biodegradation rate of PAH-degrader population due to the adaptation of the corresponding

enzymatic system So in this case the addition of a readily carbon source retards the

biodegradation process The addition of surfactant to the culture media at concentration

above their CMC is essential to increase PAH degradation rate (Pantsyrnaya et al 2011)

However Yuan et al (2000) reported negative effects when the surfactant was added at

concentration above the CMC because the excess of micelles around PAH reduces their

bioavailability (Mulligan et al 2001) However our results showed that PD and CDI were not

affected by concentrations largely beyond the CMC Some non biodegradable surfactants

can be toxic to bacteria and therefore do not improve the biodegradation process (Bautista et

al 2009) Tween-80 was the optimal surfactant for the strains of the consortium C2PL05

(Bautista et al 2009) However the optimal type of surfactant is determined by the type of

degrading strains involved in the process (Bautista et al 2009) In addition it is important to

consider the possible use of surfactant as a carbon source by the strains preferentially to

PAHs which would reduce the rates of biodegradation (Kim amp Weber 2003)

Further dilution of the inoculum represents the elimination of minority species which

could result in a decrease in the degradation ability of the consortium if the eliminated

species represented an important role in the biodegradation process (Szaboacute et al 2007)

Our results concerning the inoculum concentration showed that this factor significantly

influenced in CDI but had no effect on total PD indicating that the degrading ability of the

consortium has not been altered by the dilution of the same In Gonzalez et al (2011) the

evolution and bacterial succession of the consortium C2PL05 by culture-dependent

techniques are described All of these identified strains were efficient in degradation of PAH

(Bautista et al 2009) but Enterobacter sp was dominant at the beginning of the degradation

process whereas Stenotrophomonas sp and Pseudomonas sp were less abundant In

addition DGGE fingerprint pattern studied and described in Molina et al (2009) showed a

low microbial diversity of the consortium C2PL05 typical of an enriched consortium from

chronically contaminated soil (Vintildeas et al 2005) The results present in this work suggest

that in cultures inoculated with the highest dilution of the consortium (10-3) the less abundant

microorganisms were eliminated reducing the competition for the dominant species which

can grow vigorously

80

The influence of some environmental factors on the biodegradation of PAH can

undermine the effectiveness of the process In this study the combination of all factors

simultaneously by an orthogonal design has allowed to establish considering the interactions

between them the most influential parameters in biodegradation process Finally we

conclude that the only determining factor in biodegradation by consortium C2PL05 is the

carbon source Although cell growth is affected by temperature carbon source and inoculum

dilution these factors not condition the effectiveness of degradation Therefore the optimal

condition for a more efficient degradation by consortium C2PL05 is that the carbon source is

only PAH

Acknowledgements




Official Maacutester en Ciencia y Tecnologiacutea Ambiental of the Universidad Rey Juan Carlos


81

References


ionic surfactants on the biodegradation of PAH by diverse aerobic bacteria Int

Biodeter Biodegr 63 913-922

Boochan S Britz ML amp Stanley GA 1998 Surfactant-enhanced biodegradation of high

molecular weight polycyclic aromatic hydrocarbons by Stenotrophomonas maltophila

Biotechnol Bioeng 59 482-494

Chen S-H amp Aitken MD 1999 Salicylate stimulates the degradation of high-molecular

weight polycyclic aromatic hydrocarbons by Pseudomonas saccharophila P15

EnvironSci Technol 33 435ndash439

Chen J Wong MH Tam N 2008 Multi-factors on biodegradation kinetics of plyciclic

aromatic hydrocarbons (PAHs) by Sphingomonas sp a bacterial strain isolated from

mangrove sediment Marine Poll Bull 57 695-702

Eriksson M Ka J-O amp Mohn WW 2001 Effects of low temperature and freeze-thaw cycles

on hydrocarbon biodegradation in Artic Tundra soil Appl EnvironMicrobiol 67 5107-

5112

Gonzaacutelez N Simarro R Molina MC Bautista LF Delgado L amp Villa JA 2011 Effect of

surfactants on PAH biodegradation by a bacterial consortium and on the dynamics of

the bacterial community during the process Bioresource Technol 102 9438-9446


a bacterium isolated from Sediment below an oil field Appl EnvironMicrobiol 54

1612-1614

Jin D Jiang X Jing X amp Ou Z 2007 Effects of concenrtration head group and structure of

surfactants on the biodegradation of phenanthrene J Hazard Mater 144 215-221

Kim HS amp Weber WJ 2003 Preferential surfactant utilization by a PAH-degrading strain

effects on micellar solubilization phenomena Environ Sci Technol 37 3574-3580

Laha S amp Luthy RG 1992 Effect of non-ionic surfactants on the solubilization and

mineralization of phenanthrene in soil-water systems Biotechnol Bioeng 40 1367-

1380

Lee K Park J-W amp Ahm I-S 2003 Effect of additional carbon source on naphthalene

biodegradation by Pseudomonas putida G7 J Hazard Mater 105 157-167




Lloyd J amp Taylor JA 1994 On the temperature dependence of soil respiration Funct Ecol

8 315-323

82

Maier MR 2009 Bacterial growth In MR Maier LI Pepper and PC Gerba (Eds)

Environmental Microbiology (pp 37-54) New York Academic Press

Mohn W amp Stewart RG 2000 Limiting factors for hydrocarbon biodegradation at low

temperatures in Artic soils Soil Biol Biochem 32 1161-1172

Molina MC Gonzaacutelez N Bautista LF Sanz R Simarro R Saacutenchez I amp Sanz JL 2009



Mulligan CN Young RN amp Gibbs BF 2001 Surfactant enhanced remediation of

contaminated soil a review Eng Geol 60 371-380

Muyzer G Hottentrager S Teske A amp Wawer C 1995 Molecular microbial ecology manual

(Eds Akkermans ADL van Elsas JD Bruijn FJ) Kluwer Academic Publishers

Dordrecht pp 1-23

Pantsyrnaya T Blanchard F Delaunay S Georgen JL Geacuteudon E Guseva E amp Boudrant

J 2011 Effect of surfactants dispersion and temperature on solubility and

biodegradation of phenanthrene in aqueous media Chemosphere 83 29-33


AOF 2008 Anthracene biodegradation and surface activity by an iron-stimulated


Sartoros C Yerushalmi L Beroacuten L amp Guiot S 2005 Effects of surfactant and temperature

on biotransformation kinetics of anthracene and pyrene Chemistry 61 1042-1050


Simarro R Gonzalez N Bautista LF Sanz R amp Molina MC 2010 Optimization of key

abiotic factors of PAH (naphthalene phenanthrene and anthracene) biodegradation

process by a bacterial consortium Water Air Soil Poll 217 365-374

Sudipt KS Om VS amp Rakesh KJ 2002 Polycyclic aromatic hydrocarbons environmental

pollution and bioremediation Trends Biotechnol 20 243ndash248

Szaboacute KE Itor POB Bertilsson STranvik L amp Eiler A 2007 Importance of rare and

abundant populations for the structure and functional potential of freshwater bacterial

communities Aquatic Microbl Ecol 47 1-10

Thibault SL Anderson M amp Frankenberger WTJr 1996 Influence of surfactant on pyrene

desorption and degradation in soils Appl Environ Microbiol 62 283-287

Vintildeas M Sabateacute J Espuny MJ amp Solanas AM 2005 Bacterial community dynamics and

polycyclic aromatic hydrocarbon degradation during bioremediation of heavily

creosote-contaminated soil Appl Environ Microbiol 71 7008-7018

Wong JWC Lai KM Wan CK amp Ma KK 2000 Isolation and optimization of PAHs-

degradative bacteria from contaminated soil for PAHs bioremediation Water Air Soil

Poll 139 1-13


83

Wrenn BA amp Venosa AD 1983 Selective enumeration of aromatic and aliphatic

hydrocarbon degrading bacteria by most-probably-number (MPN) Can J Microbiol

4 252-258

Yuan SY Wei SH amp Chang BV 2000 Biodegradation of polycyclic aromatic

hydrocarbons by a mixed culture Chemosphere 41 1463-1468

Capiacutetulo

Publicado en Bioresource Technology (2011) 102 9438-9446

Gonzaacutelez N Simarro R Molina MC Bautista LF Delgado L amp Villa JA

Effect of surfactants on PAH biodegradation by a bacterial consortium

and on the dynamics of the bacterial community during the process

Efecto de los surfactantes en la biodegradacioacuten de HAP por un consorcio bacteriano y dinaacutemica de la comunidad

bacteriana durante el proceso

2

Capiacutetulo 2 Effect of surfactant on PAH biodegradation and on the dynamic of the community

87

Abstract

The aim of this work was to evaluate the effect of a non-biodegradable (Tergitol NP-10) and

a biodegradable (Tween-80) surfactant on growth degradation rate and microbial dynamics

of a polycyclic aromatic hydrocarbon (PAHs) degrading consortium (C2PL05) from a

petroleum polluted soil applying cultivable and non cultivable techniques Growth and

degradation rate were significantly lower with Tergitol NP-10 than that with Tween-80

Toxicity did not show any significant reduction with Tergitol NP-10 whereas with Tween-80

toxicity was almost depleted (30) after 40 days Regarding to the cultured bacteria

Pseudomonas and Stenotrophomonas groups were dominant during PAH degradation with

Tergitol NP-10 whereas Enterobacter and Stenotrophomonas were dominant with Tween-80

DGGE analyses (PRIMER and MDS) showed that bacteria composition was more similar

between treatments when PAHs were consumed than when PAHs concentration was still

high Community changes between treatments were a consequence of Pseudomonas sp

Sphingomonas sp Sphingobium sp and Agromonas sp


89

Introduction

Polycyclic aromatic hydrocarbons (PAH) are a group of organic pollutants composed of two

or more fused aromatic rings produced by natural and anthropogenic sources Besides

being toxic carcinogenic and mutagenic compounds the semi-volatile properties of some

PAH make them highly mobile throughout the environment (air soil and water) In addition

PAH have a high trophic transfer and biomagnification within the ecosystems due to the

lipophilic nature and the low water solubility that decreases with molecular weight (Clements

et al 1994) The importance of preventing PAH contamination and the need to remove PAH

from the environment has been recognized institutionally by the Unites States Environmental

Protection Agency (US-EPA) which has proposed 16 PAH as priority pollutants including

naphthalene phenanthrene and anthracene Currently governmental agencies scientist and

engineers have focused their efforts to identify the best methods to remove transform or

isolate these pollutants through a variety of physical chemical and biological processes

Most of these techniques involve expensive manipulation of the pollutant transferring the

problem from one site or phase to another (ie to the atmosphere in the case of cremation)

(Haritash amp Kausshik 2009) However microbial degradation is one of the most important

processes that PAH may undergo compared to others such as photolysis and volatilization

Therefore bioremediation can be an important alternative to transform PAH to less or not

hazardous forms with less input of chemicals energy and time (Haritash amp Kaushik 2009)

Most of the contaminated sites are characterized by the presence of complex mixtures

of pollutants Microorganisms are very sensitive to low concentrations of contaminants and

respond rapidly to environment perturbations (Andreoni et al 2004) Therefore microbial

communities chronically exposed to PAH tend to be dominated by those organisms capable

of use PAH as carbon and energy source (Gallego et al 2007) Even in areas previously

unpolluted there is a proportion of microbial community composed by PAH degrading

bacteria able to degrade PAH (Surridge et al 2009) These microbial communities subjected

to a polluted stress tend to be less diverse depending on the complexity of the composition

and the time of exposure (MacNaughton et al 1999) The biodegradation of hazardous

compounds by bacteria fungi and algae has been widely studied and the success of the

process will be due in part to the ability of the microbes to degrade all the complex pollutant

mixture However most of the PAH degradation studies reported in the literature have used

versatile single strains or have constructed an artificial microbial consortium showing ability

to grow with PAH as only carbon source by mixing together several known strains (Ghazali et

al 2004) Nevertheless pure cultures and synthetic microbial consortia do not represent the

natural behaviour of microbes in the environment since the cooperation among the new


90

species is altered In addition changes in microbial communities during pollutant

biotransformation processes are still not deeply studied Microbial diversity in soil

ecosystems can reach values up to 10 billion microorganisms per gram and possibly

thousands of different species although less than 10 can be culturables (Torsvik amp Ovreas

2002) Therefore additional information on biodiversity ecology dynamics and richness of

the degrading microbial community can be obtained by non-culturable techniques such as

DGGE In addition small bacteria cells are not culturable whereas large cells are supposed

to account 80 of the total bacterial volume (Nannipieri et al 2003) Therefore despite their

low proportion culturable bacteria can provide essential information about the structure and

functioning of the microbial communities With the view focused on the final bioremediation

culture-dependent techniques are necessary to obtain microorganisms with the desired

catabolic traits for bioaugmentation processes in polluted soils The PAH degradation is

limited by their low aqueous solubility but surfactants which are amphypatic molecules

enhance the solubility of hydrophobic compounds (Kolomytseva et al 2009) Previous works

(Bautista et al 2009) have shown that efficiency of a consortium named C2PL05 composed

by PAH degrading bacteria was significantly higher using surfactants

One of the main goals of the current work was to understand if culturable and non

culturable techniques are complementary to cover the full richness of a soil microbial

consortium A second purpose of the study was to describe the effect of different surfactants

(biodegradable and non-biodegradable) on growth rate PAH degradation rate and toxicity

reduction of a bacterial consortium (C2PL05) The microbial consortium C2PL05 was

isolated from a soil chronically exposed to petroleum products collected from a

petrochemical complex Finally the work is also aimed to describe the microbial dynamics

along the biodegradation process as a function of the surfactant used to increase the

bioavailability of the PAH


Chemicals and media

Naphthalene phenanthrene and anthracene (all 99 purity) were purchased from Sigmandash

Aldrich (Steinheim Germany) and Fluka (Steinheim Germany) Reagent grade

dichloromethane and n-hexane were supplied by Scharlau Chemie (Barcelona Spain)

Surfactants (Tween-80 and Tergitol NP-10) used were supplied by Sigma-Aldrich (Steinheim

Alemania) Bushnell-Haas Broth medium (BHB) was purchased from Panreac (Barcelona

Spain) and its composition is 02 g l-1 MgSO4middot7H2O 002 gmiddotl-1 CaCl2middot2H2O 10 gmiddotl-1 KHPO4


91

10 gmiddotl-1 K2HPO4 10 gmiddotl-1 NH4NO3 005 gmiddotl-1 FeCl3 Luria-Bertani media (LB) glucose and

phosphate buffer saline (PBS) were purchased from Panreac (Barcelona Spain)



Puertollano Spain To obtain the consortium 1 g of sieved soil (lt2 mm) was resuspended in

10 ml of PBS and incubated during 12 h in an orbital shaker (Innova 40 New Brunswick

Scientific Edison NJ USA) at 150 rpm and 25 ordmC under dark conditions After that 15 ml of

the supernatant was inoculated in 50 ml of BHB broth (pH 70) containing 1 wt Tween-80

as surfactant and naphthalene phenanthrene and anthracene (each at 250 mg l-1) as carbon

source Then the culture was incubated at 150 rpm and 25 ordmC under dark conditions until the

exponential phase was completed This was confirmed by monitoring the cell density by

absorbance at 600 nm in a spectrophotometer (Spectronic Genesys Thermo Fisher

Scientific Loughborough Leicestershire UK) Then the consortium was stored at 4 ordmC to

stop growth At the beginning of each experiment 500 μl of the stored consortium (asymp 16 AU)

was inoculated in Erlenmeyer flasks

Experimental design and treatments conditions

To evaluate the influence of Tween-80 and Tergitol NP-10 (biodegradable and non-

biodegradable surfactant respectively) on the degrading capacity of the consortium C2PL05

as well as the evolution of its microbial community two different treatments each in triplicate

were carried out The replicates were performed in 100 ml Erlenmeyer flasks with 50 ml of

BHB medium (pH 70) Tween-80 or Tergitol NP-10 as surfactants (1 vv) a mixture of

naphthalene phenanthrene and anthracene in n-hexane (final concentration 500 mgmiddotl-1) and

500 microl of the C2PL05 consortium (88middot108 heterotrophic cellsmiddotml-1 and 44middot107 PAH degrading

cellsmiddotml-1 for the treatments with Tween-80 73middot105 heterotrophic cellsmiddotml-1 and 24middot103 PAH

degrading cellsmiddotml-1 for treatment with Tergitol NP-10) The replicates were incubated in an

orbital shaker (Innova 40) at 150 rpm and 25 ordmC under dark conditions during 45 days

Previously to inoculate the consortium the Erlenmeyer flasks were shaked overnight to

reach the solubility equilibrium of PAH and to allow the complete evaporation of n-hexane

Samples vigorously shaking to ensure homogeneity were withdrawn twice a day for 45 days

except for the initial 24 hours where the sampling frequency was higher Cell growth PAH

(soluble and precipitated) toxicity and number of heterotrophic and PAH degrading cells


92

were measures in all samples To study the dynamic of the microbial consortium through

cultivable and non-cultivable methods samples were withdrawn at 0 15 and 30 days

Bacterial growth MPN and toxicity assays

Bacterial growth was monitored by changes in the absorbance of the culture media at 600

nm using a Spectronic Genesys spectrophotometer According to the Monod equation

(Equation 1) the specific growth rate micro is essentially equal to micromax when substrate limitation

is avoided

SK

S

S

max

(Equation 1)

Therefore from the above optical density data the maximum specific growth rate (micromax)

was estimated as the logarithmized slope of the exponential phase applying the following

equation (Equation 2)

Xdt

dX (Equation 2)

where micromax is the maximum specific growth rate Ks is the half-saturation constant S

is the substrate concentration X is the cell density t is time and micro is the specific

growth rate In order to evaluate the ability of the consortium to growth with

surfactants as only carbon source two parallel treatments were carried out at the

same conditions than the two treatments above described but in absence of PAH

Heterotrophic and PAH-degrading population from the consortium C2PL05 were

enumerated during the PAH degrading process comparing the effect of Tergitol NP-10 and

Tween-80 as surfactants The estimation was performed by using a miniaturized MPN

technique in 96-well microtiter plates with eight replicate wells per dilution Total

heterotrophic microbial population was enumerated in 180 μl of Luria Bertani (LB) medium

with glucose (15 gmiddotl-1) and 20 microl of the microbial consortium PHA-degrading population were

counted in BHB medium (180 microl including the surfactant) 20 microl of a mixture of phenanthrene

anthracene and naphthalene in hexane (each at a final concentration of 500 mgmiddotl-1) and 20 microl

of the microbial consortium in each well The MPN scores were transformed into density

estimates accounting for their corresponding dilution factors


93

The toxicity was monitored during PAH degradation and estimations were carried out

using the Microtox assay with the bioluminescent bacterium Vibrio fischeri Three controls

considered as 0 inhibition were prepared with the photobacterium and 2 NaCl (vv) and

three blanks as 100 inhibition containing only 2 NaCl (vv) Samples were salted with

NaCl (2 vv final concentration) and the toxicity was expressed as the percentage of the V

fischeri inhibition after 15 min of incubation at 15 ordmC To study the toxicity of the medium

caused by PAH when the surfactants were not added toxicity evolution was measured from

a treatment with PAH as carbon source and degrading consortia but without surfactant under

same conditions previously described

PAH monitoring

In order to compare the effect of the surfactant on the PAH depletion rate naphthalene

phenanthrene and anthracene concentrations in the culture media were analysed using a

reversed-phase C18 column (Luna C18(2) 75 cm length x 46 mm ID 3 microm particle size

Phenomenex Torrance CA USA) following the method described elsewhere (Bautista et

al 2009) The concentration of each PAH was calculated from a standard curve based on

peak area at 254 nm The apparent first-order kinetic constant (kB) due to biotic processes

was calculated by applying Equation 3

iBiiAii

i CkCkdt

dCr (Equation 3)

where C is the PAH concentration kA is the apparent first-order kinetic constant due to

abiotic processes kB is the apparent first-order kinetic constant due to biological

processes t is the time elapsed and the subscript i corresponds to each PAH



Tween-80 (1wt) naphthalene phenanthrene and anthracene (each at 500 mgmiddotl-1) without

any microbial inoculum in an orbital shaker (Innova 40) at 150 rpm and 25ordmC under dark

conditions PAH concentration in the control experiments were analyzed using the HPLC

system described previously The values of kA for each PAH were calculated by applying Eq

2 considering kB asymp 0 since no bacterial consortium was inoculated The amount of

precipitated and bioadsorbed PAH was measured after centrifugation of the samples Then

dichloromethane was added to the pellet and this extraction was repeated three times and


94

the fractions pooled The solvent was evaporated using a nitrogen flow and the extract was

dissolved into a known volume of acetonitrile for HPLC analysis

DNA extraction from cultured bacteria and phylogenetic analysis for characterization of the

PAH degrader consortium

Samples from cultures of the bacterial consortium C2PL05 during the PAH degrading

process were collected to identify the effect of the surfactants (Tergitol NP-10 and Tween-80)

To get about 20-30 colonies isolated at each collecting time samples of each treatment were

streaked onto Petri plates with BHB medium and purified agar and were sprayed with a

mixture of naphthalene phenanthrene and anthracene in n-hexane (final concentration 500

mgl-1) as carbon source The Petri plates were incubated at 25 ordmC under dark conditions

The isolated colonies were transferred onto LB agar-glucose plates in order to increase

microbial biomass for DNA extraction and stored in 50 glycerol (vv) at -80 ordmC In total 91

degrading colonies from the treatment with Tween-80 and 83 degrading colonies from the

treatment with Tergitol NP-10 were isolated

Total DNA was extracted using Microbial DNA isolation kit (MoBio Laboratories

Solano Beach CA USA) to perform the molecular identification of the PAH-degrader

isolated cultured (DIC) Amplification of the 16S rRNA coding region of the DNA was

performed as described by Vintildeas et al (2005) using the primers 16F27 (5rsquo-

AGAGTTTGATCMTGGCTCAG-3rsquo) and 16R1488 (5rsquo-TTACCTTGTTACGACTTCAGG-3rsquo) and

sequenced using the same primers Sequences were edited and assembled using

ChromasPro software version 142 (Technelysium Pty Ltd Tewantin Australia)

All of the 16S rRNA gene sequences were edited and assembled by using BioEdit

software version 487 BLAST search (Madden et al 1996) was used to find nearly identical

sequences for the 16S rRNA sequences determined Sequences were aligned using the Q-

INS-i algorithm (Katoh amp Toh 2008) of the multiple sequence alignment software MAFFT

version 6611 aligning sequences in a single step Sequence data obtained and 34

sequences downloaded from GenBank were used to perform the phylogenetic trees

Sequence divergence was computed in terms of the number of nucleotide differences per

site between of sequences according to the Jukes and Cantor algorithm (1969) The distance

matrix for all pairwise sequence combinations was analyzed with the neighbour-joining

method (NJ) of phylogenetic tree construction with 1000 bootstrap replicates by using PAUP

version 40B10 Maximum parsimony (MP) was also analyzed using PAUP 40B10 as is


95

described in Molina et al (2009) Sequences of Aquifex piruphilus were used as out-group

according to previous phylogenetic affiliations (Vintildeas et al 2005)

Denaturing gradient gel electrophoresis from microbial consortium during PAH degrading

process

Non culture dependent molecular techniques such as denaturing gradient gel

electrophoresis (DGGE) were performed to know the effect of the surfactant on the total

biodiversity of the microbial consortium C2PL05 during the PAH degradation process and

compared with the initial composition of the consortium The V3 to V5 variable regions of the

16S rRNA gene were amplified using the primers set 16S 518F and 16S 338R-GC

according to ExTaq HS DNA polymerase protocol (Promega Corp Madison WI USA)

Primers 338R-GC included a GC clamp at the 5acuteend (5acute-CGC CCG CCGCGC CCC GCG

CCC GTC CCG CCG CCC CCG CCC G-3acute) 20 μl of PCR product was loaded onto a 10

(wtvol) polyacrilamide gels that was 075 mm tick with a denaturing gradient of 35-65

(100 denaturant contained 7 M urea and 40 formamide) DGGE was performed in 1xTAE

buffer (20 M Tris-acetate 100 mM Na2EDTA pH 74) using a DGGE 2401 system (CBS

Scientific Co Del Mar CA USA) at 80 V and 60 ordmC for 16 h Gels were stained for 45 min in

1xTAE buffer containing Syber-Gold (500 μlmiddotl-1) and viewed under UV light Predominant

bands in DGGE gel were excised with a sterile razor blade and diluted in 50 μl of deionized

water overnight at 4ordmC Due to impossibility of reamplified bands DNA of the bands was

cloned in the pGEM-T Easy Vector (Promega Madison WI) Sequence of this PAH-degrader

uncultured bacterium (DUB) were edited and assembled as described above and included in

the matrix to perform the phylogenetic tree as described previously using the identification

code DUB


The maximum specific growth rate (micromax) and the kinetic constant of PAH biodegradation (kB)

were evaluated by both one and two-way analysis of variance (ANOVA) using Statistica 60

software (Statsoft Inc Tulsa OK USA) to determine differences between PAH (naphthalene

phenanthrene and anthracene) and surfactants (Tween-80 and Tergitol NP-10) Prior to

analyses Cochranrsquos C-test was used to check the assumption of homogeneity of variances

Student-Newman-Keuls test (SNK) was used to discriminate among different treatments after

significant F-test Differences in microbial assemblages were graphically evaluated for each

factor combination (surfactant and time) with non-metric multidimensional scaling (MDS)


96

using PRIMER software SIMPER method was used to identify the percent contribution of

each band to the dissimilarity or similarity in microbial assemblages between and within

combination of factors Based on Viejo (2009) bands were considered ldquohighly influentialrdquo if

they contributed to the first 70 of cumulative percentage of average dissimilaritysimilarity

betweenwithin combination of factors


Bacterial growth and toxicity media during biodegradation of PAH

Since some surfactants can be used as carbon sources cell growth of the consortium was

measured with surfactant and PAH and only with surfactant without PAH to test the ability of

consortium to degrade and grow with both surfactants (Figure 1A) The microbial consortium

C2PL05 growth was significantly lower with Tergitol NP-10 than that reached with Tween-80

which showed the best cell growth with a maximum density (Figure 1A) In addition the

growth curve with PAH and Tergitol NP-10 showed a longer latent phase (36 hours) than

with PAH and Tween-80 (lt 12 hours) The specific growth rate (micromax) of the consortium

C2PL05 was significantly higher (Table 1A) with Tween-80 than that with Tergitol NP-10 The

results showed that Tween-80 was biodegradable for consortium C2PL05 since that

surfactant was used as the only carbon source (Figure 1A) Finally when using Tergitol NP-

10 as the only carbon source growth was not observed so that this surfactant was not

considered biodegradable for the consortium

Toxicity test (bioluminescence inhibition in Vibrio fischeri) indicates that high values

observed during the PAH degrading process with Tergitol NP-10 is caused at the initial time

by both PAH and surfactant (Figure 1B) However when PAH are totally consumed (40-45

days) toxicity still remained high and constant which means that toxicity is only due to the

Tergitol NP-10 (Figure 1B) The toxicity of PAH + biodegradable surfactant (Tween-80)

treatment decreased as the PAH and the surfactant were consumed and was almost

depleted (30) after 40 days of cultivation The toxicity showed a slight increment at the

beginning of the degradation process (Figure 1B) as a consequence of the potential

accumulation of intermediate PAH degradation products (Molina et al 2009)


97

00

02

04

06

08

10

12

14

16

18

0 5 10 15 20 25 30 35 40 45

30

40

50

60

70

80

90

100

Tox

icity

(

)

Time (day)

B

A

Abs

orba

nce 60

0 nm

(A

U)

Figure 1 (A) Cell density of the consortium C2PL05 with PAH and Tween-80 () with

Tween-80 () with PAH and Tergitol NP-10() and with Tergitol NP-10 () (B)

Toxicity of the cultivation media during degradation of PAH by the consortium C2PL05

grown with Tween-80 () Tergitol NP-10 () and of the control experiment PAHs

without surfactants ()


98

The residual total concentration of three PAH of the treatments with surfactants and

the treatments without any surfactants added is shown in Figure 2 The consortium was not

able to consume the PAH when surfactants were not added PAH biodegradation by the

consortium C2PL05 was higher and faster (15 days) with Tween-80 than with Tergitol NP-10

(40 days) In all cases when surfactant was used no significant amount of PAH were

detected in precipitated or bioadsorbed form at the end of each experiment which means

that all final residual PAHs were soluble

0 5 10 15 20 25 30 35 40 45

0

10

20

30

40

50

60

70

80

90

100

Res

idua

l con

cent

ratio

n of

PA

Hs

()

Time (days)

Figure 2 Residual total PAH concentration (wt) in the treatment with Tween-80

() Tergitol NP-10 () and without surfactant ()

According to previous works (Bautista et al 2009 Molina et al 2009) these results

confirm that this consortium is adapted to grow with PAH as only carbon source and can

degrade PAH efficiently when surfactant is added According to control experiments (PAH

without consortium C2PL05) phenathrene and anthracene concentration was not affected by

any abiotic process (kA asymp 0 h-1) in the case of naphthalene some degree of abiotic depletion

was measured during the controls yielding an apparent first-order abiotic rate constant of

27middot10-3 plusmn 7middot10-5 h-1 This value was accounted for the calculation of the biodegradation rate

constant (kB) for naphthalene in the treatments so this not influence in the high

biodegradation rate of naphthalene for Tween-80 treatments The biotic depletion rate (kB) of

the three PAH was significantly higher for Tween-80 than that calculated for Tergitol NP-10

(Table 1B) There were no significant differences between PAH for Tergitol NP-10 (2 x 10-3 plusmn

4 x 10-4) whereas in the case of Tween-80 the value of kB for naphthalene (3 x 10-2 plusmn 6 x 10-4)

was higher than that for phenanthrene and anthracene (1 x 10-2 plusmn 4 x 10-4)


99

Table 1 Analysis of variance (ANOVA) for the effects of surfactants on the specific

growth rate micromax (A) and for the effects of the surfactants and PAH on the biotic

degradation rate kB (B) of the C2PL05 consortium SS is the sum of squares and df

the degrees of freedom

Effect (A) SS df F-value p-value

Surfactant 16 1 782 0001

Error 0021 2

Effect (B) SS df F-value p-value

PAH 15middot10-4 2 779 0001

Surfactant 82middot10-4 1 4042 0001

PAH x Surfactant 12middot10-4 2 624 0001

Error 203middot10-7 12

Molecular characterization of the cultured bacteria of the consortium C2PL05 and dynamics

during the PAH degradation

The identification of cultured microorganisms and their phylogenetic relationships are keys to

understand the biodegradation and ecological processes in the microbial consortia From the

consortium C2PL05 grown with Tween-80 91 strains were isolated and sequenced From

them 7 different genotypes of PAH-degrading cultures (DIC-1 JA DIC-2 JA DIC-5 JA DIC-6

JA DIC-7 JA DIC-8JA and DIC-9JA) were identified by 16S rRNA For the treatment with

Tergitol NP-10 83 strains were isolated and sequenced and 6 different genotypes were

identified (DIC-1 RS DIC-2 RS DIC-3 RS DIC-4 RS DIC-5 RS and DIC-6 JA) One of the

isolated cultures from Tergitol NP-10 showed an identical sequence to one of the strains

grown with Tween-80 therefore the previous code (DIC-6JA) was kept Table 2 show a

summary of the PAH-degrader cultures identification The aligned matrix contained 1576

unambiguous nucleotide position characters with 424 parsimony-informative Parsimony

analysis of the data matrix yielded 10 parsimonious trees with CI = 0609 and RI = 0873 In

the parsimonic consensus tree 758 of the clades were strongly supported by boostrap

values higher or equal to 70 (Figure 3) All cultivable strains identified were γ-

proteobacteria (gram-negative) and were located in three clades Pseudomonas clade

Enterobacter clade and Stenotrophomonas clade These results are consistent with those of

Vintildeas et al (2005) who observed a strong dominance of gram negative bacteria in PAH

contaminated soil during the bioremediation process In Pseudomonas clade (Figure 3) DIC

are located in three clearly groups So DIC-2RS and DIC-3RS were grouped with P

frederiksbergensis which has been previously described in polluted soils (ie Holtze et al

2006) showing ability to reduce the oxidative stress generated during the PAH degrading

process DIC-1JA DIC-2JA (Tween-80) and DIC-1RS (Tergitol NP-10) were nested in very


100

solid group characterized by the presence of the type strain P koreensis previously studied

as an agricultural soil species (Kwon et al 2003) and DIC-5RS was located in P putida

group well known by their capacity to degrade high molecular weight PAH (Samantha et al

2002) to produce surfactants (Kruijt et al 2009) and to resist high temperature and salinity

(Egamberdieva amp Kucharova 2009) So several species of Pseudomonas (ie P putida P

fluorescens) have been widely studied in bioremediation (Molina et al 2009) and the present

results confirmed that it was the most representative group with the non biodegraded

surfactant treatment DIC-7JA DIC-8JA and DIC-9JA (Tween-80) which were identified as E

cloacae (Table 2) belonged to the Enterobacter clade with a strongly statistic support (Figure

3) In this clade DIC-4RS (Tergitol NP-10) is genetically related with E ludwigii which has

been recently described as relevant medical species (Hoffman et al 2005) but completely

unknown his PAH degrading capacity Enterobacter genus has been traditionally studied by

its animal gut symbiotic function but rarely recognized as a soil PAH degrading group

(Toledo et al 2006) In this phylogenetic tree E cloacae and E ludwiggi were not resolved

This result is according to Roggenkamp (2007) who consider necessary to use more

molecular markers within Enterobacter taxonomical group in order to contrast the

phylogenetic relationships In addition Enterobacter genera may not be a monophyletic

group (Kampfer et al 2005) Therefore more phylogenetic studies need to be done to clarify

the species concept within this group Finally DIC-5JA (Tween-80) and DIC-6JA isolated

from experiments using both surfactants (Tween-80 and Tergitol NP) are clearly belong to

type strain Stenotrophomonas clade genetically close to S maltophiliaT (Table 2) which has

been described as PAH-degrader (Zocca et al 2004)


101

Figure 3 Neighbour joining tree showing the phylogenetic relationship of the 16S rRNA for the PAH-

degrader isolated culture (DIC) from the consortium C2PL05 with Tergitol NP (DIC-1JA ndash DIC-9JA)

and Tween-80 (DIC-1RS ndash DIC-5RS) and PAH-degrader uncultured bacteria (DUB) obtained from

DGGE of the consortium with both surfactant at 0 15 and 30 days of the process Boostrap values of

neighbourjoining and parsimonus higher than 50 are showed on the branch of the tree (NJMP) No

incongruence between parsimony and neighbour joining topology were detected Pseudomonas

genus has been designated as P Pantoea genus as Pa Sphingobium as S and Sphingomonas as

Sp Xantomonas as X and Xyxella as Xy T= type strain


102

Table 2 Bacteria identification and percentage of similarity from the GenBank data base Degrading

uncultured bacteria (DUB) form DGGE bands and degrading isolates cultured (DIC)

Colonies identified by cultivable techniques

DIC simil Mayor relationship with bacteria

of GenBank(acc No) Phylogenetic group

DIC-1RSb 980 Pseudomonas koreensis (NR025228) Pseudomonadaceae (γ)

DIC-2RS b 1000 Pseudomonas frederiksbergensis (AY785733)

Pseudomonadaceae (γ)



DIC-4RS b 990 Enterobacter ludwigii (AJ853891) Enterobacter cloacae (EU733519)

Enterobacteriaceae (γ)

DIC-5RS b 990 Pseudomonas putida (EU046322) Pseudomonadaceae (γ) DIC-6JA-6RS c 1000 Stenotrophomonas maltophilia (AY512625) Xanthomonadaceae (γ) DIC-1JA a 9900 Pseudomonas koreensis (NR025228) Pseudomonadaceae (γ) DIC-2JA a 9900 Pseudomonas koreensis (NR025228) Pseudomonadaceae (γ) DIC-5JA a 9964 Stenotrophomonas maltophilia (AY512625) Xanthomonadaceae (γ) DIC-7JA a 9985 Enterobacter cloacae (AF157695) Enterobacteriaceae (γ) DIC-8JA a 9993 Enterobacter cloacae (AF157695) Enterobacteriaceae (γ) DIC-9JA a 100 Enterobacter cloacae (AF157695) Enterobacteriaceae (γ)

Identification by non-cultivable techniques

DUB Band

simil Mayor relationship with bacteria

of GenBank (acc No) Phylogenetic group

DUB-1RS a 1 970 Uncultured Nitrobacteria sp (AM990004) Caulobacteraceae (α) DUB-2RS b 21 980 Bradyrhizobium sp (HQ171485) Bradyrhizobiaceae (α) DUB-3RS b 24 940 Uncultured bacterium (AY939443) -- DUB-4RS b 24 100 Uncultured Pseudomonas sp (HM561497) Pseudomonadaceae (γ) DUB-10RSb 28 980 Uncultured Sphingomonas sp (HM438638) Sphingomonadaceae(α) DUB-11RSa 28 960 Pseudomonas sp (HM468085) Pseudomonadaceae (γ) DUB-6RS b 29 980 Pseudomonas stutzeri (HQ130335) Pseudomonadaceae (γ) DUB-8RS b 29 980 Pseudomonas sp (HM468085) Pseudomonadaceae (γ) DUB-5RS b 29 990 Sphingobium sp (EF534725) Sphingomonadaceae(α) DUB-7RS b 29 980 Sphingobium sp (EF534725) Sphingomonadaceae(α) DUB-9RS b 30 970 Bacterium (AJ295668) --

a DIC or DUB belonging to treatments with Tween-80 b DIC or DUB belonging to treatments with Tergitol NP-10 c DIC or DUB belonging to treatments with Tween-80 and Tergitol NP-10

With respect to the dynamics of the microorganisms isolated from the microbial

consortium C2PL05 (Figure 4) Pseudomonas (DIC-1RS DIC-2RS and DIC-5RS Figure 4A

4B) with a percentage around 60 and Stenotrophomonas (only DIC-6JA Figure 4A and

4D) with presence of 90 were dominant groups during the PAH degrading process with

Tergitol NP-10 at 0 and 30 days in the case of Pseudomonas and at 15 days in the case of

Stenothrophomonas Enterobacter (DIC-4RS Figure 4A 4D) only was present at the end of

the process with a percentage around 40 With Tergitol NP-10 Pseudomonas sp group

was dominant coincident with the highest relative contribution of PAH degrading bacteria to


103

total heterotrophic bacteria at the beginning (33 of contribution) and at the end of the

degradation process (41) However Enterobacter (DIC-7JA DIC-8JA and DIC-9JA Figure

4E and 4H) with a maximum presence of 98 at 0 days and Stenotrophomonas (DIC-6JA

Figure 4E and 4G) with a maximum presence of 85 at the end of the process were

dominant with the biodegradable Tween-80 Thus Enterobacter sp seems to start the PAH

degradation process and Stenotrophomonas to finish it but at 15 days three groups coexist

within a contribution ranging 20 to 50 (Figure 4E) Therefore in agreement with other

authors (Colores et al 2000) the results of the present work confirm changes in the

bacterial (cultured and non-cultured) consortium succession during the PAH degrading

process driven by surfactant effects According to Allen et al (1999) the diversity of the

bacteria cellular walls may explain the different tolerance to grow depending on the

surfactant used Previous works (Piskonen amp Itaumlvaara 2004) have shown the capacity of

some bacteria to use both surfactants (Tergitol NP-10 and Tween-80) as carbon sources

However in agreement with recent studies (Bautista et al 2009) the present work confirms

that Tergitol NP-10 is not degradable by the consortium C2PL05 These results showed a

drastic change of the consortium composition after the addition of surfactant


104

0 15 30

0102030405060708090

100

102030405060708090

100

D

C

B

A

0 15 30

F DIC-1JA DIC-2JA

E

G DIC-6JA DIC-5JA

0 15 30

H

Time (day)

DIC-7JA DIC-8JA DIC-9JA

Pse

udom

onas

ribot

ypes

(

)

DIC-1RS DIC-2RS DIC-3RS DIC-5RS

102030405060708090

100

Ste

notr

opho

mon

as

ribot

ypes

(

)

DIC-6JA

0 15 30

102030405060708090

100

Ent

erob

acte

r rib

otyp

es (

)

DIC-4RS

Time (days)

Tot

al s

trai

ns (

)

Figure 4 (A) Dynamics of the microbial consortium C2PL05 during PAH degradation process with

Tergitol NP-10 and (E) with Tween-80 as surfactants Isolated and identified genus were

Pseudomonas () Stenotrophomonas () and Enterobacter () Dynamics and succession of

the (B) Pseudomonas (C) Stenotrophomonas and (D) Enterobacter ribotypes with Tergitol NP-10

as surfactant Dynamics and succession of the (F) Pseudomonas (G) Stenotrophomonas and (H)

Enterobacter ribotypes


105

Biodiversity and evolution of the non-cultivable bacteria of the consortium during PAH

degradation

The most influential DGGE bands to similarity 70 of contribution according to the results of

PRIMER analyses were cloned and identified allowing to know the bands and species

responsible of similarities and dissimilarities SIMPER procedure (Clarke 1993) was used to

identify the percentage contribution () that each band made to the measures of the Bray-

Curtis similarity between treatments at each surfactant (Tween-80 and Tergitol-NP) and time

(initial time after 15 and 30 days) Bands were selected as lsquoimportantrsquo to be identified if they

contributed to the first 70 of cumulative percentage of average similarity between

treatments Summary of the identification process are shown in Table 2 Phylogenetic

relationship of these degrading uncultured bacteria was included in the previous

parsymonious tree (Figure 3) In total 11 uncultured bacteria were identified DUB-4RS

DUB-6RS DUB-8RS and DUB-11RS were located in the Pseudomonas clade but these

uncultured bacteria were no grouped with a particular species of the genus DUB-5RS DUB-

7RS were identified as Sphingobium sp and DUB-10RS as Sphingomonas sp and located

in the Sphingobium and Sphingomonas clade respectivelly DUB-2RS was nested in

Bradyrhizobium clade because was identified as Bradyrhizobium sp and this clade was

supported by the type strain B japonicum In the same way DUB-1RS identified as

Uncultured Nitrobacteria was located in the Nitrobacteria clade belonged to N

hamadeniensis type strain Finally DUB-3RS and DUB-9RS were not identified with a

particular genus so they were located in a clade composed by uncultured bacteria The

phylogenetic relationship of these degrading uncultured bacteria allows expanding

knowledge about the consortium composition and process development Some of them

belong to α-proteobacteria DUB-5RS and DUB-7RS were related to Sphingobium group and

DUB-10RS with Sphingomonas clade thought this relationship should be confirmed

considering the low boostrap values Sphingomonas is a genus frequently isolated as PAH

degrader (Jing et al 2007) and important in the degradation of phenanthrene metabolites

(Tao et al 2007) Similarly Sphingobium sp has been described as PAH degrader

specifically in phenanthrene degradation process (Jing et al 2007) DUB-2RS belonged to

Bradyrhizobiaceae phylogenetic group and although Bradyrhizobium are genera barely

described as PAH degrading bacteria some studies based on PAH degradation by chemical

oxidation and biodegradation process have described that this plant-associated bacteria are

involved in the degradation of extracting agent used in PAH biodegradation techniques in

soils (Rafin et al 2009) DUB-1RS is a genotype related to Nitrobacteria clade However

Nitrobacteria has not been described as PAH degrader but this bacteria transform nitrites in

nitrates from the oxidation of nitrites (Modrogan et al 2010) and it is likely involved in the


106

nitrites oxidation process when the bioavailability of PAH in the media are low and so it is

not toxic for this bacteria Finally DUB-8RS DUB-6RS and DUB-11RS showed a high

similitude with Pseudomonas strain though the phylogenetic relationship with Pseudomonas

clade of DUB-11RS should be confirmed

Analysis of DGGE gel (Figure 5) showed that treatment with Tergitol NP-10 had very

few changes during biodegradation process whereas when the consortium was grown with

the biodegradable surfactant Tween-80 more changes were observed Similarity (Table 3)

between treatments were compared and analyzed by type of surfactant (Tween-80 vs

Tergitol NP-10) or by sampling time (15 days vs 30 days) The MDS analysis (Table 3)

showed the lowest values of Bray Curtis similarity coefficient between the consortium at

initial time (T0 not exposed to PAH and surfactants) with the PAH and Tween-80 after 15

days (16) and 30 days (7) However the similarity of T0 with PAH and Tergitol NP-10 after 15

days (22) and 30 days (26) was much higher Similarity between time treatments (15 and 30

days) within Tergitol NP-10 (56) was higher than with Tween-80 (32) The similarity within

treatments with Tween-80 was mainly due to the bands 1 and 29 (Table 3 Uncultured

Nitrobacteria and Sphingobium and Pseudomonas respectively see Table 2) whereas the

similarity within Tergitol NP-10 treatment was due to bands 1 and 30 (Table 3 Uncultured

Nitrobacteria and Uncultured bacteria respectively see Table 2)


107

Figure 5 Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA gen fragments

from the consortium C2PL05 with initial inoculum (lane 1) with Tween-80 at 15 (lane 2) and 30 (lane 4)

days with Tergitol NP-10 at 15 (lane 3) and 30 (lane 5) days and molecular weight markers (lane 0)

According to PRIMER analysis similar bands between treatments (15 and 30 days) with Tergitol NP-

10 () and between treatments (15 and 30 days) with Tween-80 () are shown

1 Uncultured Nitrobacteria sp(DUB-1RS) 21 Bradyrhizobium sp (DUB-2RS) 24 Uncultured bacterium (DUB-3RS)

Uncultured Pseudomonas (DUB-4RS) 28 Uncultured Sphingomonas sp (DUB-10RS)

Pseudomonas sp (DUB-11RS) 29 Pseudomonas stutzeri (DUB-6RS) Pseudomonas sp

(DUB-8RS) Sphingobium sp(DUB-5RS DUB-7RS)

30 Uncultured Bacterium (DUB-9RS)


108

Table 3 Bands contributing to approximately the first 70 of cumulative percentage

of average similarity () Bands were grouped by surfactant and time

Band Tween-80 TergitolNP-10 15 days 30 days 1 3828 2105 2707 3014 29 2969 1509

30 2469 19

24 881 3447

27 845

21 516

Cumulative similarity () 3168 4479 4479 3391 Cloning was not possible

The genera identified in this work have been previously described as capable to

degrade the three PAH completely and efficiently with a reduction of the toxicity (Bautista et

al 2009) In the case of the treatment with Tween-80 the lower biodiversity may be caused

by a few dominant species of these genera driven during the PAH degradation process by

antagonist and synergic bacterial interactions and not by differences in the functional

capacities However when consortium grows with a non-biodegradable surfactant there is

higher biodiversity of species and interaction because the activity of various functional

groups can be required to deal the unfavorable environmental conditions

Conclusions

The choice of surfactants to increase bioavailability of pollutants is critical for in situ

bioremediation because toxicity can persist when surfactants are not biodegraded

Nevertheless surfactants affect the dynamics of microbial populations in a stable PAH-

degrading consortium From the application point of view the combination of culturable and

non culturable identification techniques may let to optimize the bioremediation process For

bioaugmentation processes culturable tools help to select the more appropriate bacteria

allowing growing enough biomass before adding to the environment However for

biostimulation process it is important to know the complete consortium composition to

enhance their natural activities


109

Acknowledgment

Authors are deeply indebted to Raquel Sanz Laura Saacutenchez and Laura Garciacutea and for their

support during the development of the experiments Authors also gratefully acknowledged

the financial support from the Spanish Ministry of Environment (Research project 1320062-

11) and Fundacioacuten Alfonso Martiacuten Escudero Authors also thank Repsol-YPF for providing

the soil samples This work is framed within the Official Maacutester en Ciencia y Tecnologiacutea

Ambiental from Universidad Rey Juan Carlos


110

References

Allen CRC Boyd DR Hempenstall F Larkin MJ amp Sharma D 1999 Contrasting effects

of a nonionic surfactant on the biotransformation of polycyclic aromatic hydrocarbons

to cis-dihydrodiols by soil bacteria Appl Environ Microbiol 65 1335-1339

Andreoni V Cavalca L Rao MA Nocerino G Bernasconi S DellrsquoAmico E Colombo M

amp Gianfreda L 2004 Bacterial communities and enzyme activities of PAH polluted

soils Chemosphere 57 401-412



Biodeter Biodegr 30 1ndash10

Clements WH Oris JT amp Wissing TE 1994 Accumulation and food chain transfer of

fluoranthene and benzo[a]pyrene in Chironomus riparius and Lepomis macrochirus

Archiv Environ Contam Toxicol 26 261ndash266

Colores GM Macur RE Ward DM amp Inskeep WP 2000 Molecular analysis of

surfactant-driven microbial population shifts in hydrcarbon-contaminated soil Appl

Environ Microbiol 66 2959-2964

Egamberdieva D A amp Kucharova Z 2009 Selection for root colonising bacteria stimulating

wheat growth in saline soils Biol Fert Soils 45 563ndash571

Gallego RJS Garciacutea-Martiacutenez MJ Llamas JF Belloch C Pelaacuteez AI amp Saacutenchez J

2007 Biodegradation of oil tank bottom sludge using microbial consortia

Biodegradation 18 269ndash281

Ghazali FM Rahman RNZA Salleh AB amp Basri M 2004 Degradation of hydrocarbons

in soil by microbial consortium Int Biodeter Biodegr 54 61ndash67

Haritash AK amp Kaushik CP 2009 Biodegradation aspects of Polycyclic Aromatic

Hydrocarbons (PAH) A review J Hazard Mater 169 1-15

Hoffmann H Stindl S Stumpf A Mehlen A Monget D Heesemann J Schleifer KH amp

Roggenkamp A 2005 Description of Enterobacter ludwigii sp Nov a novel

Enterobacter species of clinical relevance Syst Appl Microbiol 28 206ndash212

Holtze MS Nielsen P Ekelund F Rasmussen LD amp Johnsen K 2006 Mercury affects

the distribution of culturable species of Pseudomonas Appl Soil Ecol 31 228ndash238

Jing W Hongke X amp Shaohui G 2007 Isolation and characterization of a microbial

consortium for effectively degrading phenanthrene Pet Sci 4 68-75

Jukes TH amp Cantor R 1969 Evolution of protein molecules in Mammalian protein

metabolism (H Munro ed) Academic Press New York


111

Katoh K amp Toh H 2008 Improved accuracy of multiple ncRNA alignment by incorporating

structural information into a MAFFT-based framework BMC Bioinformatics 9 paper

212

Kampfer P Ruppel S amp Remus R 2005 Enterobacter radicincitans sp Nov a plant

growth promoting species of the family Enterobactriaceae Syst Appl Microbiol 28

213ndash221

Kolomytseva MP Randazzo D Baskunov BP Scozzafava A Briganti F amp Ludmila A

2009 Role of surfactants in optimizing fluorene assimilation and intermediate

formation by Rhodococcus rhodochrous VKM B-2469 Bioresource Technol 100

839-844

Kruijt M Tran H amp Raaijmakers JM 2009 Functional genetic and chemical

characterization of biosurfactants produced by plant growth-promoting Pseudomonas

putida J Appl Microbiol 107 546-556

Kwon SW Jong WS Kim S Park IC Yoon SH Park DH Lim CK amp Go SJ 2003

Pseudomonas koreensis sp Nov Pseudomonas umsongensis sp Nov and

Pseudomonas jinjuensis sp Nov novel species from farm soils in Korea Int J Syst

Evol Microbiol 53 21ndash27

Laha S amp Luthy RG 1991 Inhibition of phenantrene mineralization by nonionic surfactants

in soil-water systems Environ Sci Technol 25 1920-1930

Madden TL Tatusov RL amp Zhang J 1996 Applications of network BLAST server Method

Enzymol 266 131-141 (available at URL httpncbinlmnihgovBLAST)

MacNaughton SJ Stephen JR Venosa AD Davis GA Chang Y amp White DC 1999

Microbial population changes during bioremediation of an experimental oil spill Appl


Modrogan C Diaconu E Orbulet OD amp Miron AR 2010 Forecasting Study for Nitrate Ion

Removal Using Reactive Barriers Rev Chim 6 580-584




Nannipieri P Ascher J Ceccherini MT Landi L Pietramellara G amp Renella G 2003

Microbial diversity and soil functions Eur J Soil Sci 54 655-670

Piskonen R amp Itaumlvaara M 2004 Evaluation of chemical pretreatment of contaminated soil

for improved PAH bioremediation Appl Microbiol Biotechnol 65 627-634

Rafin C Veignie E Fayeulle A amp Surpateanu G 2009 Benzo[a]pyrene degradation using

simultaneously combined chemical oxidation biotreatment with Fusarium solani and

cyclodextrins Bioresource Technol 100 3157-3160

Roggenkamp A 2007 Phylogenetic analysis of enteric species of the family

Enterobacteriaceae using the oric-iocus Syst Appl Microbiol 30 180-188


112

Samantha SK Singh OV amp Jain RK 2002 Polycyclic aromatic hydrocarbons

environmental pollution and bioremediation Trends Biotechnol 20 243-248

Surridge AKJ Wehner FC amp Cloete TE 2009 Bioremediation of Polluted Soil in Singh

A Kuhad RC Ward OP (Eds) Adv Appl Biorem p 103-121 Springer Berlin

Tao X-Q Lu G-N Dang Z Yi X-Y amp Yang C 2007 Isolation of ohenanthrene-degrading

bacteria and characterization of phenanthrene metabolites Worl J Biotechnol 23

647-6554

Toledo FL Calvo C Rodelas B amp Gonzaacutelez-Loacutepez J 2006 Selection and identification of

bacteria isolated from waste crude oil with polycyclic aromatic hydrocarbons removal

capacities Syst Appl Microbiol 29 244ndash252

Torsvik V amp Ovreas L 2002 Microbial diversity and function in soil from genes to

ecosystems Curr Opin Microbiol 5 240ndash245

Viejo RM 2009 Resilience in intertidal rocky shore assemblages across the stress gradient

created by emersion times Mar Eco- Prog Ser 390 55-65




Zocca C Gregori SD VisentiniF amp Vallini G 2004 Biodiversity amongst cultivable

polycyclic aromatic hydrocarbon-transforming bacteria isolated from an abandoned

industrial site FEMS Microbiol Lett 238 375-382

Capiacutetulo

Enviado a FEMS Microbiology Ecology en Diciembre 2012

Simarro R Gonzaacutelez N Bautista LF amp Molina MC


bacterial consortium at low temperatures

Biodegradacioacuten de hidrocarburos de alto peso molecular por un consorcio bacteriano

degradador de madera a bajas temperaturas

3

Capiacutetulo 3 HMW-PAH biodegradation by a wood bacterial consortium at low temperatures

115

Abstract

The aim of this work was to evaluate the ability of two bacterial consortia (C2PL05 and

BOS08) extracted from very different environments to degrade low (naphthalene

phenanthrene anthracene) and high (pyrene and perylene) molecular weight polycyclic

aromatic hydrocarbons (PAH) at high (15-25ordmC) and low (5-15ordmC) temperature ranges

C2PL05 was isolated from a soil in an area chronically and heavily contaminated with

petroleum hydrocarbons and BOS08 from decomposing wood in an unpolluted forest free of

PAH Bacterial consortia were described by cultivable and no-cultivable techniques (DGGE)

PAH-degrading bacterial population measured by most probable number (MPN)

enumeration increased during the exponential phase Toxicity estimated by MicrotoxTM

method was reduced to low levels and the final PAH depletion determined by high-

performance liquid chromatography (HPLC) confirmed the high degree of low and high

molecular weight PAH degradation capacity of both consortia The PAH degrading capacity

was also confirmed at low temperatures and specially by consortium BOS08 where strains

of Acinetobacter sp Pseudomonas sp Ralstonia sp and Microbacterium sp were identified


117

Introcuduction

Polycyclic aromatic hydrocarbons (PAH) constitute a diverse class of organic compounds

formed by two or more aromatic rings in several structural configurations having

carcinogenic mutagenic and toxic properties Therefore environment contamination by PAH

is currently a problem of concern and it has been shown that bioremediation is the most

efficient practice retrieving the original conditions of the ecosystem (Haritash amp Kaushik

2009) However the high molecular weight PAH (HMW-PAH) such as pyrene

benzo[a]pyrene or benzo[b]fluoranthene are generally recalcitrant and resistant to microbial

attack due to their low solubility and bioavailability Therefore these compounds are highly

persistent in the environment and bioaccumulated in organisms (Lafortune et al 2009)

Studies on PAH biodegradation with less than three rings have been the subject of many

reviews (ie Sutherland et al 1995) However there is a lack of knowledge about the

HMWndashPAH biodegradation (Kanaly amp Harayama 2000)

Microbial communities play an important role in the biological removal of pollutants in

soils (MacNaughton et al 1999) Therefore changes in environmental condition may alter

species diversity of the soil microbiota and their metabolic rates (Margesin amp Schinner

2001) In areas chronically polluted by PAH there are abundant bacteria able to degrade

those toxic contaminants by using them as sole carbon and energy sources (Taketani et al

2010) Recent works (Tian et al 2008 Surridge et al 2009 Couling et al 2010) have

reported the potential ability to degrade PAH by microorganisms apparently not previously

exposed to those toxic compounds This is extensively known for lignin degrading white rot-

fungi that produce a set of extracellular enzymes such as oxidases and peroxidases (Wong

2009) with low substrate specificity that expand their oxidative action beyond lignin being

capable to degrade other complex phenolic compounds and PAH (ie Canet et al 2001)

Although less extensively than in fungus PAH degradation capacity have been also reported

in this type of environment in bacteria belonged to genera Pseudomonas (Zimmermann

1990 McMahon et al 2007) However according to Couling et al (2010) the wide-spread

capacity to degrade PAH by microbial communities even from unpolluted soils can be

explained by the fact that PAH are ubiquitously distributed by natural process throughout the

environment at low concentration enough for bacteria to develop degrading capacity

Regardless of these issues there are some abiotic factors such as temperature that

may greatly influence biodegradation process It has been shown (Mohn amp Stewart 2000)

that although biodegradation of PAH is more efficient in the range 20-30 ordmC it can be carried

out even in colder (lt5 ordmC) environments (Eriksson et al 2001) At low temperature diffusion


118

and solution rates and so bioavailability of PAH decreases (Haritash amp Kaushik 2009)

Simultaneously the microbial metabolism is slowed-down increasing the lag period (Atlas amp

Bartha 1972 Eriksson et al 2001) However and according to the hypothesis that

degrading microorganisms are present in most of ecosystems there are degrading bacteria

adapted to low temperatures (Yakimov et al 2003 Brakstad amp Bonaunet 2006) that can

express degrading capacity So the study of biodegradation at low temperatures is important

since the temperature of more than 90 of the seawater volume is below 5 ordmC In addition

PAH and anthropic discharges sometimes have occurred and may occur in sea water (Bode

et al 2006 Soriano et al 2006) or in cold and even extreme environments such as in

Alaska (Bence et al 1996)

The main goal of this work was to study the effect of low temperature on HMW-PAH

degradation rate by two different consortia isolated from two different environments one from

decay wood in an unpolluted forest (consortium BOS08) and other from a polluted soil

exposed to hydrocarbons The purpose of the present work was also to describe the

microbial dynamics along the biodegradation process as a function of temperature and type

of consortium used


Chemicals and media

Naphthalene phenanthrene anthracene pyrene and perylene (all gt99 purity) purchased

from Sigma-Aldrich (Steinheim Germany) and Fluka (Steinheim Germany) were prepared

in a stock solution in n-hexane (Fluka Steinheim Germany) to get a final concentration of

002 gl-1 for naphthalene phenanthrene and anthracene 001 gl-1 for pyrene and 0005 gl -1

for perylene Tween-80 purchased from Sigma-Aldrich was added according to previously

work (Bautista et al 2009) Composition of optimized Bushnell Haas Broth medium (BHB)

(Simarro et al 2010) was 02 g l -1 MgSO4middot7H2O 002 g l -1 CaCl2 2H2O 0088 g l -1 KHPO4

0088 g l -1 K2HPO4 209 g l -1 NaNO3 0015 g l -1 Fe2(SO4)3

Physicochemical characterization of soils and isolation of bacterial consortia

Consortia C2PL05 was isolated from a permanently polluted soil from a petroleum refinery

(Ciudad Real Spain) with a range of environmental temperatures from 10 ordmC in winter to 25

ordmC in summer The consortium BOS08 was extracted from dead wood in a pristine Atlantic


119

forest in Fragas do Eume Galicia Spain (latitude 43ordm 4175acute north longitude 8ordm 0683acute west)

with oaks as the dominant flora species and with a range of temperatures of 10 ordmC in winter

and 18 ordmC in summer To obtain the microbial consortia sieved soil and the wood sample

were suspended in PBS (110) and stirred overnight at 25 ordmC Then 15 ml of each extract

was inoculated in 50 ml of BHB (pH 70) with Tween-80 1 (vv) as surfactant and

naphthalene phenanthrene anthracene pyrene and perylene (each at 500 mg l -1) as carbon

sources Each culture were incubated in an orbital shaker at 150 rpm 25 ordmC and dark

conditions until the exponential phase was completed (asymp 5 days) monitoring cell density by


Scientific Loughborough Leicestershire UK)

Sieved (lt2 mm) river sand was used as substrate Prior to use it was burned at 550

ordmC in a furnace to remove organic matter and microorganisms Water holding capacity (WHC)

of the river sand was measured following the method described by Wilke (2005)


15 microcosms (triplicates by five different incubation times) were performed with consortium

C2PL05 at high temperature range (H) 16 hours with light at 25 ordmC followed by 8 hours in

the dark at 15 ordmC Another 15 microcosms with consortium C2PL05 were incubated at low

temperature range (L) 16 hours with light at 15 ordmC followed by 8 hours in the dark at 5 ordmC

The same experiments were performed with consortium BOS08 Microcosms were incubated

in suitable chambers equipped with temperature lightdarkness cycle and humidity (60)

control systems Each microcosms contained 90 g of sterilized sand 18 ml of BHB (60 of

WHC) with Tween-80 1 (vv) 2 ml of PAH stock solution in n-hexane (final amount of PAH

per tray of 20 mg of naphthalene 20 mg of phenanthrene 20 mg of anthracene 10 mg of

pyrene and 5 mg of perylene) and 35 ml of bacterial consortium (0088 AU = 275x104

cellsmiddotml -1 for C2PL05 and 0051 AU = 286 x 104 cellsmiddotml-1 for BOS08)


Bacterial density during the PAH degrading process was monitored at 0 11 33 66 101 and

137 days by changes in the absorbance of the culture media at 600 nm in a

spectrophotometer (Spectronic GenesysTM England) From the absorbance data the

intrinsic growth rate in the exponential phase was calculated by applying Equation 1


120

1

1

ii

iii tt



corresponds to each sample or sampling time Increments were normalized by

absorbance measurements at initial time (day 0) to correct the inoculum dilution effect

Heterotrophic and PAH-degrading population from the consortia were estimated by a

miniaturized most probably number technique (MPN) in 96-well microtiter plates with eight

replicate wells per dilution (Wrenn amp Venosa 1996) Total heterotrophic microbial population

was estimated in 180 μl of Luria Bertani (LB) medium with glucose (15 gl -1) and 20 microl of the

microbial consortium PAH-degrading population in the inoculum was estimated in 180 microl of

BHB medium containing Tween-80 (1 vv) 10 microl of PAH stock mix solution as only carbon

source (n-hexane was allowed to evaporate prior to inoculation) and 20 microl of the microbial

consortium in each well

Toxicity during the PAH degradation was also monitored through screening analysis of

the samples following the MicrotoxTM method with the luminescent bacterium Vibrio fischeri

following the protocol suggested by Microbics Corporation (1992) The toxicity was

expressed as the percentage of the V fischeri inhibition after 15 min of incubation at 15 ordmC

Monitoring of PAH biodegradation

To confirm that consortium BOS08 was not previously exposed to PAH samples were

extracted with acetone and n-hexane according to Joslashrgensen et al (2005) and the

identification was performed by GC-MS analysis of the extract A gas chromatograph (model

CP3800 Varian Palo Alto CA USA) equipped with a Varian Factor Four VF-1ms capillary

column (15 m length 025 mm ID 025 μm film thickness) was coupled to a quadruple

mass-spectrometer detector (Model 1200L Varian) The stationary phase was composed by

phenyl (5) and dimethylpolysiloxane (95) as carried gas ice in the mobile phase

Temperature gradient program used was initial temperature of 80 ordmC for 2 min temperature

increase to 300 ordmC at the rate of 1 ordmC min-1 final temperature of 300 ordmC for 15 min with a

final duration of the method of 39 min In addition total petroleum hydrocarbons (TPH) in

both soils were extracted and quantified as is described previously

PAH from microcosms were extracted and analyzed at initial and final time to estimate

the total percentage of PAH depletion by gas cromatography using the gas cromatograph


121

equiped and protocol described previuosly For this 100 g of soil from each replicate were

dried overnight at room temperature and PAH were extracted with 100 ml of dichloromethane

during 3 hours in a Soxhlet device The solvent was removed in a rotary evaporator and the

residue was dissolved in 1 ml of dichloromethane an encapsulated in a vial to inyect 05 μl in

the FDI chromatograph



To identify cultivable microorganisms samples from each microcosm were collected at zero

33 and 101 days of the biodegradation process To extract the microorganisms 15 g of soil

were suspended in PBS (110) and incubated overnight in an orbital shaker at 150 rpm

maintaining the same temperature and light conditions than during the incubation process

To get about 10 PAH-degrading colonies isolated 100 ml of the supernatant were placed

onto Petri plates with BHB medium and purified agar and were sprayed with a stock mix

solution of naphthalene phenanthrene anthracene pyrene and perylene (final concentration

500 mgL-1) as carbon source and incubated at the same temperature conditions

Total DNA of the PAH-degrading isolated cultures (DIC) was extracted using Microbial

DNA kit (MoBio Laboratories Solano Beach CA USA) and amplified using primers 16S F27

and 16S R1488 (Lane et al 1991) according to the ExTaq HS DNA polymerase protocol

(Molina et al 2009) Sequences were edited and assembled using ChromasPro software

version 142 (Technelysium Pty Ltd Tewantin Australia) to check for reading errors and

when possible resolving ambiguities BLAST search (Madden et al 1996 available at URL

httpncbinlmnihgovBLAST) was used to find nearly identical sequences for the 16S

rRNA sequences determined Sequences were aligned using the Q-INS-i algorithm (Katoh amp

Toh 2008a) of the multiple sequence alignment software MAFFT version 6611 (Katoh amp

Toh 2008b) aligning sequences in a single step

All identified sequence (by culture and no-culture techniques) and more similar

sequences downloaded from GenBank were used to perform the phylogenetic tree




method (NJ) of phylogenetic tree construction with 1000 bootstrap replicates using PAUP

40B10 (Swofford 2003) In addition maximum parsimony (MP) was also analyzed (Molina


122

et al 2009) Sequences of Aquifex piruphilus and Hydrogenobacter hydrogenophylus were

used as out-group

Denaturing gradient gel electrophoresis (DGGE) from microbial consortia during PAH

degrading process

A non culture-dependent molecular techniques as DGGE was performed to know the effect

of the temperature on total biodiversity of both microbial consortia during the PAH

degradation process by comparing the treatment at zero 33 and 101 day with the initial

composition of the consortia Total DNA was extracted from 025 g of the samples using

Microbial Power DNA isolation kit (MoBio Laboratories Solano Beach CA USA) and

amplified using the primers set 16S 338F-GC and 16S 518R according to ExTaq HS DNA

polymerase protocol (Promega Corp Madison WI USA) PCR product was loaded onto a

10 (wv) polyacrilamide gel with a denaturing gradient from 35 to 65 denaturant Gel

were stained with Syber-Gold and viewed under UV light and predominant bands in DGGE

gel were excised Due to impossibility to reamplify bands DNA of the bands was cloned in

the pGEM-T Easy Vector (Promega Madison WI) PAH-degrader uncultured bacterium

(DUB) were edited and assembled as described above and included in the matrix to perform

the phylogenetic tree Images of DGGE were digitalized and processed using the UN-Scan-It

gel analysis software version 60 (Silk Scientific US)

To identifiy the presence of fungi in the consortium BOS08 during the process total

DNA was extracted from the samples using Microbial Power DNA isolation kit (MoBio

Laboratories Solano Beach CA USA) and amplified with the primers set 18S ITS1F and

ITS4 according to Quiagen Multiplex PCR kit protocol DNA of Clitocybe metachroa was

extracted using DNeasy Plant Mini Kit (Quiagen) from the mushroom for use as PCR

positive PCR products were visualized under UV light on an agarose gel (1 ) using Syber-

Gold as intercalating agent


In order to evaluate the effects of inocula type and temperature on the final percentage of

PAH depletion and on the intrinsic growth rate (μ) bifactorial analysis of variance (ANOVA)

were used The variances were checked for homogeneity by the Cochranacutes test Student-

Newman-Keuls (SNK) test was used to discriminate among different treatments after

significant F-test representing this difference by letters in the graphs Data were considered


123

significant when p-value was lt 005 All tests were done with the software Statistica 60 for

Windows Differences in microbial assemblages were graphically evaluated for each factor

combination (time type of consortium and temperature) with a non-metric multidimensional

scaling (MDS) using PRIMER software (Clarke 1993) SIMPER method was used to identify

the percent contribution of each band from DGGE to the dissimilarity or similarity in microbial

assemblages between and within combination of factors Based on Viejo (2009) bands were

considered ldquohighly influentialrdquo if they contributed to the first 60 of cumulative percentage of

average dissimilaritysimilarity betweenwithin combination of factors

Results

Hydrocarbons in soils

Figure 1 shows GC-MS analysis of the extracted hydrocarbons from samples where both

consortia were isolated Soil samples where C2PL05 consortium was isolated contained 64

wt of total petroleum hydrocarbons (TPH) However no traces of PAH or any other

petroleum hydrocarbons were detected within samples where BOS08 consortium was

obtained

0 5 10 15 20 25 30 35

BO S08

C 2PL05

tim e (m in)

Figure 1 GC-MS total ion chromatogram from solvent extracted hydrocarbons in soils where

consortia C2PL05 and BOS08 were isolated


124

Cell growth intrinsic growth MPN and toxicity assays

Figure 2 (A B) shows the growth of both microbial consortia during PAH biodegradation

process Lag phases were absent and long exponential phases (until day 66 approximately)

were observed in all treatments except with the C2PL05 consortium at low temperature

(finished at day 11) In general higher cell densities were achieved in those microcosms

incubated in the higher temperature range Despite similar cell densities reached with both

consortia and both temperature levels the values of the intrinsic growth rate (μ) during the

exponential phase (Table 1) showed significant differences between consortia and

temperatures of incubation but not in their interaction (Table 2A) Differences between

treatments showed that the highest μ was obtained at high temperatures (25 ordmC-15 ordmC) and

with BOS08 consortium

Figure 2 (C D) showed that the initial number of PAH-degrading bacteria were at least

one order of magnitude lower than heterotrophic bacteria in both consortia The highest

heterotrophic bacteria concentration was reached after 33 days of incubation approximately

to a final value of 108- 109 cells g-1 soil (four orders of magnitude above the initial values)

The highest contribution of PAH-degrading bacteria to total heterotrophic bacteria was

observed at 33 days of incubation No differences were observed between temperature

ranges From 33 days both type of populations started to decrease but PAH-degrading

bacteria of consortia increased again at 101 days reaching values at the end of the process

similar to the initial ones


125

0 11 33 66 101 137

005

010

015

020

025

030

035

0 11 33 66 101 137

0 33 101 137102

103

104

105

106

107

108

109

0 33 101 137Time (day)Time (day)

Time (day)

Abs

orba

nce 6

00nm

(A

U)

Time (day)

DC

BA

cell

g so

il

Figure 2 Cell growth of consortia C2PL05 (A) and BOS08 (B) at high () and low () temperature

range during PAH biodegradation and MPN for consortia C2PL05 (C) and BOS08 (D) of heterotrophic

(squares) and PAH-degrading (circles) cultivated at high (filled symbols) and low (empty symbols)

temperature range


126

Table 1 Intrinsic growth rate (μ) and biodegradation percentage of phenanthrene (Phe) anthracene

(Ant) pyrene (Pyr) perilene (Per) and total PAH (Tot) at final time for consortia C2PL05 and BOS08 at

high (H) and low (L) temperature range Superscript letters (a to c) show differences between groups

(plt005 SNK) and plusmn SD the standard deviation

μ

Treatment d-1x10-3 plusmnSD x10-3

C2PL05 H 158 b 09 C2PL05 L 105 a 17

BOS08 H 241 c 17

BOS08 L 189 b 12

PAH biodegradation ()

Treatment Phe plusmn SD Ant plusmn SD Pyr plusmn SD Per plusmn SD Total plusmn SD

C2PL05 H 954 plusmn 04 993 plusmn 01 270 plusmn 62 986 plusmn 01 989 c plusmn 04

C2PL05 L 801 plusmn 61 459 plusmn 158 470 plusmn 118 538 plusmn 190 543 a plusmn 109

BOS08 H 938 plusmn 27 993 plusmn 04 472 plusmn 130 864 plusmn 61 866 bc plusmn 60

BOS08 L 940 plusmn 31 579 plusmn 31 542 plusmn 102 691 plusmn 137 677 ab plusmn 77

Table 2 Analysis of variance (ANOVA) of the effects on μ (A) total PAH biodegradation (B) and

biodegradation of pyrene and perilene (C) SS is the sum of squares and df the degree of freedoms

Factor df SS F

p-value

A) μ

Temperature a 1 36 x 10-3 5931 Consortium b 1 83 x 10-5 136

Temperature x Consortium 1 20 x 10-4 343 ns

Error 8 49 x 10-5 0001

B) Total PAH biodegradation ()

Treatment c 3 3526 73

Error 8 1281

C) Biodegradation of pyrene and perilene ()

Treatment c 3 11249 11 ns

PAH d 1 85098 251

Treatment x PAH 3 31949 31 ns

Error 16 54225

a high (15-25ordmC) or low (5-15ordmC) temperature range b consortium C2PL05 or BOS08 c C2PL05 at

high and temperature range or BOS08 at high and low temperature range d naphthalene

phenanthrene anthracene pyrene and perylene p lt 005 p lt 001 p lt 0001


127

With regard to toxicity values (Figure 3) complete detoxification were achieved at the

end of each treatment except for consortium C2PL05 (percentage of toxicity 40) incubated

at low temperature (Figure 3A) When consortium BOS08 was incubated at low temperature

there was a time period between 11 and 66 days that toxicity increased (Figure 3B)

0 11 33 66 101 137

0

20

40

60

80

100

0 11 33 66 101 137

BA

Time (day)

Tox

icity

(

)

Time (day)

Figure 3 Toxicity of microcosms with consortium C2PL05 (A) and BOS08 (B) incubated at high ()

and low () temperature range during PAH biodegradation process

Biodegradation of PAH

PAH biodegradation results are shown in Table 1 PAH depletion showed significantly

differences (Table 2B) within the consortium C2PL05 with highest values at high temperature

and the lowest at low temperature (Table 1) Those differences were not observed within the

BOS08 consortium and PAH depletion showed average values between values of C2PL05

depletion Regarding each individual PAH naphthalene was completely degraded at final

time 80 of phenanthrene was depleted in all treatments and anthracene and perylene

were further reduced at high (gt85) rather than low temperature (gt50) However pyrene

was significantly less consumed by the consortia than perylene (Table 1 and Table 2C)

Phylogenetic analyses

Phylogenetic relationships of the degrading isolated cultures and degrading uncultured

bacteria are shown in Figure 4 The aligned matrix contains 1349 unambiguous nucleotide

position characters with 505 parsimony-informative and 173 characters excluded Parsimony


128

analysis of the data matrix yielded 87 parsimonious trees with CI = 0756 RI = 0945 and a

length of 1096 Figure 4 also shows the topology of the neighbour joining tree

Inconsistencies were not found when analysing boostrap values of neighbour joining (NJ)

and maximum parsimony (MP)


degrader isolated culture (DIC) and degrading uncultured bacteria (DUB) obtained from DGGE of the

consortia and cultivable identification process at day 0 33 and Boostrap values of neighbour joining

(NJ) and parsimonious (MP) are showed on the branch of the tree (NJMP) No incongruence between

parsimony and neighbour joining topology were detected Pseudomonas genus has been designated

as P Psychrobacter genus as Ps Acinetobacter as A and Ralstonia as R T= type strain


129

DIC-46-RS (Rhodococcus sp) DIC-47RS (Bacillus psychrodurans) and DUB-25RS

(Microbacterium sp) were not included in the phylogenetic tree due to their high phylogenetic

distance with most of the DIC and DUB Phylogenetic tree was composed by bacteria

belonged to γ- and β-Proteobacteria Group of γ- Proteobacteria was composed by

Acinetobacter clade Psychrobacter clade and Pseudomonas clade whereas β-

Proteobacteria group was only composed by Ralstonia clade Within Acinetobacter clade

although the identity approximation (BLAST option Genbank) reported A johnsonii and A

haemolyicus such as the species closest to some of the DIC and DUB the incorporation of

the types strains in the phylogenetic tree species do not showed a clear monophyletic group

Thus and as a restriction molecular identification of these strains (Table 3) was exclusively

restricted to genus level that is Actinobacter sp A similar criteria was taken for

Pseudomonas clade where molecular identifications carry out through BLAST were not

supported by the monophyletic hypothesis when type strains were included in the analysis

Psycrobacter clade sister group of Acinetobacter clade are represented by Psychrobacter

urativorans type strain (DQ143924T) in which DIC-14RS and DIC-23RS are nested β-

Proteobacteria is only composed by Ralstonia clade confirmed by RinsidiosaT (FJ772078T)

although DICs included in this clade are more related with the strain Ralsonia sp AF488779


130

Table 3 Identification and similarity () to bacteria from GenBank of cultivable strains

and DGGE bands (non-cultivable bacteria)

Days Consortium Temperature Strains Molecular Identification

(genera) 33

C2PL05

15 ordmC-5 ordmC

DIC-7RS DIC-8RS DIC-9RS DIC-11RS DIC-10RS DIC-12RS DUB-25RS DUB-26RS

Acinetobacter sp Acinetobacter sp Acinetobacter sp Acinetobacter sp Acinetobacter sp Acinetobacter sp Microbacterium sp Acinetobacter sp

25 ordmC-15 ordmC

DIC-13RS DIC-14RS DIC-15RS DIC-46RS DUB-24RS DUB-25RS DUB-26RS

Acinetobacter sp Psychrobacter urativorans Pseudomonas sp Rhodococcus sp Pseudomonas sp Microbacterium sp Acinetobacter sp

BOS08

15 ordmC-5 ordmC

DIC-16RS DIC-17RS DIC-18RS DIC-19RS DIC-20RS DIC-21RS DIC-22RS DUB-25RS DUB-26RS

Ralstonia sp Ralstonia sp Ralstonia sp Ralstonia sp Ralstonia sp Ralstonia sp Ralstonia sp Microbacterium sp Acinetobacter sp

25 ordmC-15 ordmC

DIC-23RS DIC-47RS DUB-22RS DUB-23RS DUB-24RS DUB-25RS DUB-26RS

Psychrobacter urativorans Bacillus psychrodurans Pseudomonas sp Pseudomonas sp Pseudomonas sp Microbacterium sp Acinetobacter sp

101

C2PL05

15ordmC-5ordmC

DIC-24RS DIC-25RS DIC-26RS DIC-27RS DUB-25RS DUB-26RS

Acinetobacter sp Acinetobacter sp Pseudomonas sp Pseudomonas sp Microbacterium sp Acinetobacter sp

25 ordmC-15 ordmC

DIC-28RS DIC-29RS DIC-30RS DIC-31RS DIC-32RS DUB-24RS DUB-25RS DUB-26RS

Acinetobacter sp Acinetobacter sp Acinetobacter sp Pseudomonas sp Pseudomonas sp Pseudomonas sp Microbacterium sp Acinetobacter sp

BOS08

15 ordmC-5 ordmC


Acinetobacter sp Acinetobacter sp Acinetobacter sp Acinetobacter sp Acinetobacter sp Acinetobacter sp Acinetobacter sp Microbacterium sp Acinetobacter sp


131

25 ordmC-15 ordmC


Acinetobacter sp Acinetobacter sp Acinetobacter sp Acinetobacter sp Pseudomonas sp Pseudomonas sp Microbacterium sp Acinetobacter sp

Diversity and evolution of cultivated and uncultivated bacteria and dynamics during PAH

biodegradation

PCR analysis to identify fungal DNA in BOS08 was negative for the initial period of the

biodegradation process at both temperatures ranges Fungal DNA was only positive at high

temperatures and the end of the biodegradation process (101 and 137 days)

A minimum of 10 colonies were isolated and molecularly identified from the four

treatments at days 33 and 101 by cultivated methods The most influential bands of DGGE

to 60 of contribution to similarity (Figure 5 Table 4) according to the results of PRIMER

analysis were cloned and identified with the except of bands 2 4 27 and 36 that were not

cloned after several attempts likely due to DNA degradation The results of the identification

by cultivated and uncultivated methods (Table 3 Figure 5) show that different strains of

Acinetobacter (DUB-26RS uncultured Acinetobacter sp) and Microbacterium bands 24

(DUB-26RS uncultured Acinetobacter sp) and 22 (DUB-25RS Microbaterium sp)

respectively were always present in both consortia (Figure 5) both at high and low

temperatures However it should be also noted that Rhodococcus sp strains are unique to

C2PL05 consortium whereas Ralstonia sp and Bacillus sp were only found in BOS08

consortium being all of the above DIC strains (Table 3) In depth analysis of the community

of microorganisms through DGGE fingerprints and further identification of the bands allowed

to establish those bands responsible for the similarities between treatments (Table 4) and the

most influential factor MDS (Figure 6) shows that both time and temperature have and

important effects on C2PL05 microbial diversity whereas only time had effect on BOS08

consortium Both consortia tend to equal their microbial compositions as the exposed time

increase (Figure 6) The highest average of similarity (5327 ) was observed at day 101

being bands 36 4 (unidentified) and 24 (DUB-26RS Acinetobacter sp) responsible for that

similarity The lowest similarity (3543 ) was observed within the consortium C2PL05 (Table

4) being the high abundance of the band 20 (DUB-24RS Pseudomonas sp) and the lack of

the band 22 (DUB-25R Microbacterium sp) responsible of the dissimilarity Concluding it

can be observed that bands 20 (DUB-24RS Pseudomonas sp) 22 (DUB-25R


132

Microbacterium sp) 24 (DUB-26RS Acinetobacter sp) 36 and 4 (both unidentified) were

the most responsible for the similarity or dissimilarity between bacterial communities of

different treatments Another band showing lower contribution to these percentages but yet

cloned was band 12 from which two genotypes were identified (DUB-22RS and DUB-23RS)

as Pseudomonas sp Regarding to identification of DIC strains (Table 3) Rhodococcus sp

was exclusive of C2PL05 consortium and Ralstonia sp and Bacillus sp were only found in

BOS08 consortium

Table 4 Bands contribution to 60 similarity primer between treatments grouped by time type

of bacterial consortium and incubation temperature Average similarity of the groups determine

by SIMPER method

Time (day) Consortium Temperature

Band DUB 0 33 101 C2PL0 BOS0 High Low

22 DUB-25RS 2855 2789 2581 20 DUB-24RS 2993 2521 1797 2366

36 Unidentified 3546 1029 210

4 Unidentified 2855 1120 2362 1755 2315 175

27 Unidentified 139

2 Unidentified 1198

24 DUB-26RS 929

Cumulative similarity () 5710 5781 5595 6081 6134 5710 524Average similarity () 4433 4070 5327 3543 4660 4433 405

Unidentified bands from DGGE after several attempts to clone


133

Figure 5 Denaturant gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA gen

fragments from the consortium C2PL05 (line 1 to 5) and consortium BOS08 (line 6 to 10) Line 0

contains the molecular weight markers lines 1 and 6 correspond to initial time lines 2 and 7 to

high temperature range at day 33 lines 3 and 8 to high temperature range at day 101 lines 4

and 9 to low temperature range at day 33 and lines 5 and 10 to low temperature range at day

101


134

Figure 6 Multidimensional scaling (MDS) plot showing the similarity

between consortia BOS08 (BO) and C2PL05 (C2) incubated at low

(superscript L) and high (superscript H) temperature at day 0 33 and

101(subscripts 0 1 and 2 respectively)

Discussion

PAH degradation capability of bacterial consortia

Consortium BOS08 was isolated from a pristine soil since hydrocarbons and especially PAH

were not detected Opposite results were observed for samples where consortium C2PL05

was extracted with a large amount (64 wt) of petroleum compounds (ltC40) measured

However both consortia proved to be able to efficiently degrade HMW-PAH even at low

temperature range (5-15 ordmC) However both consortia have shown lower pyrene than

perylene depletion rates despite the former has lower molecular size and higher aqueous

solubility and bioavailability and lower hydrophobicity Previous work (Alves et al 2005)

have reported that UV and visible light can activate the chemical structure of some PAH

inducing changes in toxicity However whereas these authors classified phototoxicity of

pyrene and perylene as positive other phototoxic classifications (Mekenyan et al 1994)

consider pyrene as extremely toxic and perylene as moderately toxic So the high toxicity

level of pyrene due to phototoxic effect may explain the lower depletion rates of pyrene

opposite to that expected from their physicochemical properties above mentioned

Contrary to previous works (Spain amp ven Veld 1983 Jhonsen amp Karlson 2005) the

consortium BOS08 has not needed the pre-exposure to PAH to induce microbial adaptation


135

and consequently degradation of those pollutants In agreement with previous works

(Margesin amp Schinner 2001) our results have showed that the addition of PAH to the forest

consortium BOS08 was rapidly followed by an initial increase of PAH degrading bacteria

Considering the origin of consortium BOS08 extracted from a soil rich in organic matter and

decaying wood is possible that biodegradation process may be associated with wood

degrading bacteria and fungi However results confirmed that initial conditions when PAH

concentration was high fungi were not present Fungi appeared just at the end of the

biodegradation process (101 and 137 days) and only at high temperature when high PAH

concentration was already depleted and toxicity was low These results therefore confirm

that biodegradation process was mainly carried out by bacteria when PAH concentration and

toxicity were high

PAH degradation ability is a general characteristic present in some microbial

communities when community is exposed to PAH (Macleod amp Semple 2002 Jhonsen amp

Karlson 2005 Tian et al 2008) Microbial consortia were obtained from highly different

levels of contamination However although high differences were observed at the initial

microbial composition of both consortia they share some strains (Microbacterium sp and

Acinetobacter sp) The lower diversity found within the C2PL05 consortium (more details in

Molina et al 2009) obtained from a chronically and heavily polluted area with petroleum

hydrocarbons is typical of aged soils exposed to PAH These pollutants drive the selection of

specific bacteria that are able to degrade them (Vintildeas et al 2005)

Most of the identified species by DGGE (culture-independent rRNA approaches) in this

work were γ-proteobacteria (Pseudomonas and Acinetobacter) except DUB-26RS 98

similar to Microbacterium sp belonging to Actinobacteria phylum In agreement with previous

works (Harayama et al 2004) identification results retrieved by culture-dependent methods

showed some differences from those identified by the culture-independent rRNA

approaches DIC identified by culturable techniques belonged to a greater extend to

Proteobacteria phylum γ-Proteobacteria (Pseudomonas Pshycrobacter Acinetobacter) and

β-Proteobacteria (Ralstonia) Only two cultivable strains DIC-46RS and DIC-47RS identified

as Rhodococcus sp and Bacillus psychrodurans belonged to Actinobacteria and Firmicutes

phylum respectively Genera as Bacillus Pseudomonas and Ralstonia were identified within

the consortium BOS08 obtained from decaying wood in a pristine forest These genera are

typical from decomposing wood systems and have been previously mentioned as important

aerobic cellulose-degrading bacteria such as Bacillus sp (Lynd et al 2002) or degraders of

the highly oxidized oxalate (Pseudomonas sp Ralstonia sp) which is released by white-rot

fungi during degradation of lignocellulose (Dutton amp Evans 1996) Lignin is one of the most

slowly degraded components of dead plants and the major contributor to the formation of


136

humus as it decomposes The breakdown of lignin is mediated by extracellular enzymes

such as laccase lignin peroxidise and manganese peroxidase (Hatakka 1994 Hatakka

2001) The lack of specificity and the high oxidant activity of these enzymes make them able

to degrade different components as PAH (ie Pickard 1999) For this reason Bacillus

Pseudomonas and particularly Ralstonia identified within the consortium BOS08 and

typical from decomposing wood systems have been also previously identified as degrader of

aromatic compounds (Zhuang et al 2002 Chauhan et al 2008 Luo et al 2009) While

many eukaryotic laccases have been identified and studied laccase activity has been

reported in relatively few bacteria these include some strains identified in our decomposing

wood consortium BOS08 such as Ralstonia sp and Bacillus sp and others like Azospirillum

lipoferum Marinomonas mediterranea Amycolatopsis sp Streptomyces coelicolor

Arthrobacter cholorophenolicus and Rhodococcus opacus (McMahon et al 2007 Dawkar et

al 2009 Brown et al 2011)

HMW-PAH degradation at low temperatures

In the last 10 years research in regard to HMW-PAH biodegradation has been carried out

mainly through single bacterial strains or artificial microbial consortia and at optimal

temperatures (Kanaly amp Harayama 2000 Kanaly amp Harayama 2010) However there is a

lack of knowledge focused on HMW-PAH biodegradation at difficult conditions such as low

temperatures by full microbial consortia Temperature is a key factor in physicochemical

properties of PAH and in the control of PAH biodegradation metabolism in microorganisms

The diffusion rate of PAH into the aqueous phase increases with temperature and so PAH

bioavailability (Haritash amp Kaushik 2009) and PAH metabolism rate (Leahy amp Colwell 1990)

In agreement with previous results (Eriksson et al 2001) PAH biodegradation rates were

significantly higher at moderate temperatures (15-25 ordmC) because metabolic activity

diffusion and mass transfer was facilitated However there are also microorganisms with

capacity to efficiently degrade HMW-PAH even at lower temperatures (Margesin et al 2002)

as microorganisms present at both consortia (BOS08 and C2PL05)

Genera as Acinetobacter and Pseudomonas identified from both consortia growing at

low temperature have been previously reported as typical strains from cold and petroleum-

contaminated sites being capable to grow using solely hydrocarbons (MacCormack amp Fraile

1997 Eriksson et al 2003 Margesin et al 2003) According to previously works that

considered this genera as cold-tolerant (Margesin et al 2003 Ma et al 2006) our results

showed that they grow and efficiently degrade HMW-PAH at low temperature range (5-15 ordmC)

but with significantly lower rates than those at higher temperature In addition whereas time


137

was an influence factor in bacterial communities distribution temperature only affected to

C2PL05 consortium Possibly these results can be related with the environmental

temperature of the sites where consortia were extracted Whereas bacterial community of

BOS08 are adapted to temperatures below 20 ordmC all year C2PL05 consortium is adapted to

a range with maximum temperatures above 20 ordmC Hence although this consortium had cold-

tolerant species that degrade at low temperatures their probably less proportion than in the

BOS08 consortium resulted in differences between percentages of PAH depletion and

evolution of the bacterial community in function of temperature Therefore the cold-adapted

microorganisms are important for the in-situ biodegradation in cold environments

Acknowledgements

This work has been funded by the Spanish Ministery of Enviroment (Projects 11-37320053-

B and 0120062-11) and by Fundacioacuten Alfonso Martiacuten Escudero


138

References

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Can J Microbiol 18 1851-1855


ionic surfactants on the biodegradation of PAH by diverse aerobic bacteria Int Biodet

Biodegr 63 913-922

Bence AE Kvenvolden KA amp Kennicutt MC 1996 Organic geochemistry applied to

environmental assessments of Prince William Sound Alaska after Exxon Valdez oil

spill-a review Org Geochem 24 7-42

Bode A Gonzaacutelez N Lorenzo J Valencia J Varela MM amp Varela M 2006 Enhanced

bacterioplankton activity after the Prestige oil spill off Galicia NW Spain Aquatic

Microb Ecol 43 33-41

Brakstad OG amp Bonaunet K 2006 Biodegradation of petroleum hydrocarbons in seawater

at low temperatures (0ndash5degC) and bacterial communities associated with degradation

Biodegradation 17 71-82

Brown ME Walker MC Nakashige TG Iavarone AT amp Chang M 2011 Discovery and

characterization of heme enzymes from unsequenced bacteria Application to microbial

lignin degradation J Am Chem Soc 4518006-18009

Canet R Birnstingl JG Malcolm DG Lopez-Real JM amp Beck AJ 2001 Biodegradation

of polycyclic aromatic hydrocarbons (PAH) by native microflora and combinations of

white-rot fungi in a coal-tar contaminated soil Bioresource Technol 76113-117

Chauhan A Fazlurrahman Oakeshot JG amp Jain RK 2008 Bacterial metabolism of

polycyclic aromatic hydrocarbons strategies for bioremediation Indian J Microbiol 48

95-113

Clarke KR 1993 Non-parametric multivariate analyses of changes in community structure

Aust Ecol 18 117-143

Couling NR Towel MG amp Semple KT 2010 Biodegradation of PAH in soil Influence of

chemical structure concentration and multiple amendment Environ Pollut 158 3411-

3420

Dawkar VV Jadhav UU Telke AA amp Govindwar SP 2009 Peroxidase from Bacillus sp

VUS and its role in the decolorization of textile dyes Biotechnol Bioprocess Eng 14

361-368

Dutton MV amp Evans CS 1996 Oxalate production by fungi its role in pathogenicity and

ecology in the soil environment Can J Microbiol 42 881ndash895


139

Eriksson M Jong-Ok Ka amp Mohn WW 2001 Effects of low temperature and freeze-thaw

cycles on hydrocarbon biodegradation in Arctic Tundra soil Appl Environ Microbiol

675107-5112

Eriksson M Sodersten E Yu Z Dalhammar G amp Mohn WW 2003 Degradation of

polycyclic aromatic hydrocarbons at low temperature under aerobic and nitrate-

reducing conditions in enrichment cultures from northern soils Appl Environ

Microbiol 69 275-84

Harayama S Kasai Y amp Hara A 2004 Microbial communities in oil-contaminated seawater

Curr Opin Biotechnol 15 205-214

Haritash AK amp Kaushik CP 2009 Biodegradation aspects of Polycoilyclic aromatic

hidrocarbons (PAH) A review J Hazard Mater 169 1-15

Hatakka A 1994 Lignin-modifying enzymes from selected white rot fungi production and

role in lignin degradation FEMS Microb Rev 13 125-135

Hatakka A 2001 Biodegradation of lignin In Hofrichter M Steinbuchel A(eds)

Biopolymers vol 1 Lignin humic substances and coal Wiley-VCH Weinheim

Germany p129-180

Johonsen A amp Karlson U 2005 PAH degradation capacity of soil microbial communities-

does it depend on PAH exposure Microb Ecol 50 488ndash495

Joslashrgensen KS Jaumlrvinen O Sainio P Salminen J amp Suortti AM 2005 Quantification of

soil contamination In Margesin R Schinner F (eds) Manual of soil analysis

monitoring and assessing soil bioremediation Springer Berlin pp 97-119

Kanaly RA amp Harayama S 2000 Biodegradation of high-molecular-weight polycyclic

aromatic hydrocarbons by bacteria J Bacteriol 182 2059ndash2067

Kanaly RA amp Harayama S 2010 Advances in the field of high-molecular-weight polycyclic

aromatic hydrocarbon biodegradation by bacteria Microb Biotechnol 3 136ndash164

Katoh K amp Toh H 2008a Recent developments in the MAFFT multiple sequence alignment

program Brief Bioinform 9 286ndash298

Katoh K amp Toh H 2008b Improved accuracy of multiple ncRNA alignment by incorporating

structural information into a MAFFT-based framework BMF Bioinform 9 212

Lafortune I Juteau P Deacuteziel E Leacutepine F Beaudet R amp Villemur R 2009 Bacterial

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Lane DJ 1991 16S23S sequencing In E Stackebrandt and M Goodfellow (ed) Nucleic

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Leahy JG amp Colwell RR 1990 Microbial degradation of hydrocarbons in the environments

Microbiol Rev 54 305-315


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Luo YR Tian Y Huang X Yan CL Hong HS Lin GH amp Zheng TL 2009 Analysis of

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by DGGE Marine Poll Bull 58 1159-1163

Lynd LR Weimer PJ van Zyl WH amp Pretorius IS 2002 Microbial cellulose utilization

fundamentals and biotechnology Microbiol Mol Biol Rev 66 506ndash577

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Macleod CJA amp Semple KT 2002 The adaptation of two similar soils to pyrene catabolism

Environ Pollut 119357-364

MacNaughton SJ Stephen JR Venosa AD Davis GA Chang YJ amp White DC 1999



Madden TL Tatusov RL Zhang J 1996 Applications of network BLAST server Method


Margesin R amp Schinner F 2001 Bioremediation (natural attenuation and biostimulation) of

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Margesin R Zacke G amp Schinner F 2002 Characterization of heterotrophic

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Margesin R Gander S Zacke G Gounot AM amp Schinner F 2003 Hydrocarbon

degradation and enzyme activities of cold-adapted bacteria and yeasts Extremophiles

7451ndash458

McMahon AM Doyle EM Brooksm S amp OacuteConnor KE 2007 Biochemical

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Pseudomonas putida F6 Enzyme Microb Tech 401435-1441

Mekenyan OG Ankly GT Veith GD amp Call DJ 1994 QSAR for photoinduced toxicity I

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Mohn WW amp Stewart GR 2000 Limiting factors for hydrocarbon biodegradation at low

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Pickard MA Roman R Tinoco R Vazquez-Duhalt R 1999 Polycyclic aromatic

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Spain JC amp vanVeld PA 1983 Adaptation of natural microbial communities to degradation

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Surridge AKJ Wehner FC amp Cloete TE 2009 Bioremediation of Polluted Soil In Singh

A Kuhad RC Ward OP (eds) Adv Appl Biorem 103-121 Springer Berliacuten

Sutherland JB Rafii F Khan AA amp Cerniglia CE 1995 Mechanisms of polycyclic

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Swofford DL 2003 PAUP Phylogenetic analysis using parsimony (and another methods)

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Vintildeas M Sabateacute J Espuny MJ Solanas AM 2005 Bacterial community dynamics and



Tian Y Luo Y Zheng T Cai L Cao X amp Yan C 2008 Contamination and potential

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Taketani RG Franco NO Rosado AS amp van Elsas JD 2010 Microbial community

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Wilke BM 2005 Determination of chemical and physical soil properties In Margesin R

Schinner F (eds) Manual of soil analysis monitoring and assessing soil

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Wong WSD 2009 Structure and action of ligninolytic enzymes Appl Biochem Biotechnol

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hydrocarbon-degrading bacterial by most probably number (MPN) Can J Microbiol

42 252-258


142

Yakimov MM Giuliano L Gentile G Crisafi E Chernikova TN Abraham W-R Luumlnsdorf

H Timmis KN amp Golyshin PN 2003 Oleispira antarctica gen nov sp nov a novel

hydrocarbonoclastic marine bacterium isolated from Antarctic coastal sea water Int J

System Evol Microbiol 53779-785

Zhuang W-Q Tay J-H Maszenan AM amp Tay STL 2002 Bacillus naphthovorans spnov

from oil contaminated tropical marine sediments and its role in naphthalene

biodegradation ApplMicrobiol Biotechnol 58547-553

Zimmermann W 1990 Degradation of lignin by bacteria J Biotechnol 13119-130

Proteobacteria

Capiacutetulo


Simarro R Gonzaacutelez N Bautista LF Molina MC Peacuterez M amp Peacuterez L

Assessment the efficient of bioremediation techniques (biostimulation bioaugmentation

and natural attenuation) in a creosote polluted soil change in bacterial community

Evaluacioacuten de la eficacia de teacutecnicas de biorremediacioacuten (bioestimulacioacuten bioaumento y

atenuacioacuten natural) en un suelo contaminado con creosota cambios en la comunidad bacteriana

4

Capiacutetulo 4 Efficient of biorremediation techniques in creosote polluted soil

145

Abstract

The aim of the present work was to assess different bioremediation treatments

(bioaugmentation bioestimulation combination of both and natural attenuation) applied to a

creosote polluted soil with a purpose of determine the most effective technique in removal of

pollutant Toxicity microbial respiration degradation of creosote and PAH (antharcene

phenathrene and pyrene) as well as evolution of bacterial communities by non culture-

dependent molecular technique DGGE were analyzed Results showed that creosote was

degraded through time without significant differences between treatments but PAH were

better degraded by treatment with biostimulation Low temperatures at which the process

was developed negatively conditioned the degradation rates and microbial metabolism as

show our results DGGE results revealed that biostimulated treatment displayed the highest

microbial biodiversity However at the end of the bioremediation process no treatment

showed a similar community to autochthonous consortium The degrader uncultured bacteria

identified belonged to Pseudomonas Sphingomonas Flexibacter all of them involved in

degradation process Particularly interesting was the identification of two uncultured bacteria

belonged to genera Pantoea and Balneimonas did not previously describe as such


147

Introduction

Creosote is a persistent chemical compound derived from burning carbons as coal between

900-1200 ordmC and has been used as a wood preservative It is composed of approximately

85 polycyclic aromatic hydrocarbons (PAH) 10 phenolic compounds and 5 nitrogen

and sulfur PAH are a class of fused-aromatic compounds toxic mutagenic bioaccumulative

and persistent in the environment and so the United State Environmental Protection Agency

(US EPA) considered that the removal of these compounds is important and priority Against

physical and chemical methods bioremediation is the most effective versatile and

economical technique to eliminate PAH Microbial degradation is the main process in natural

decontamination and in the biological removal of pollutants in soils chronically contaminated

(MacNaughton et al 1999) in which degrading-bacteria are abundant (Taketani et al

2010) However recently works have reported (Tian et al 2008 Couling et al 2010) the

potential ability to degrade PAH of microorganisms from soils apparently not exposed

previously to those toxic compounds The technique based on this degradation capacity of

indigenous bacteria is the natural attenuation This technique avoid damage in the habitat

(Dowty et al 2001) allowing to retrieve the original conditions of the ecosystem converting

the toxic compounds into harmless (Kaplan amp Kitts 2004 Haritash amp Kaushik 2009)

However this method require a long period or time to remove the toxic components because

the number of degrading microorganisms in soils only represents about 10 of the total

population (Yu et al 2005a) Many of the bioremediation studies are focused on the

bioaugmentation which consist in the inoculation of allochthonous degrading

microorganisms (ie Atagana 2006) However bioaugmentation is a complicate technique

to study because a negative or positive effect depends on the interaction between the

inocula and the indigenous population due to the competition for resources mainly nutrients

(Yu et al 2005b) Other bioremediation techniques as biostimulation consist in to empower

the degrading capacity of the indigenous community by the addition of nutrients to avoid

metabolic limitations (ie Vintildeas et al 2005)

However inconsistent results have been reported with all these previuos treatments

Previous studies have shown that biodegradation rates can be increased (Mills et al 2004)

and have no effect influenced negatively with biostimulation (Yu et al 2005a Chen et al

2008) Similar enhance of biodegradation rates (Atagana et al 2006) and not significant

differences (Vintildeas et al 2005 Yu et al 2005b) have been described with bioaugmentation

It is necessary taking in to account that each contaminated site can respond in a different

way (Vintildeas et al 2005) therefore to carry out an in situ bioremediation process it will be

necessary to design a laboratory-scale assays to determine what technique is more efficient


148

on the biodegradation process and the effect on the microbial diversity In addition

previously works (Gonzalez et al 2011) showed that although PAH were completely

consumed by microorganisms toxicity values remained above the threshold of the non-

toxicity Although most of the work not perform toxicity assays these are necessary to

determine effectiveness of a biodegradation The main goal of the present study is to

determine through a laboratory-scale assays the most effective bioremediation technique in

decontamination of creosote contaminated soil evaluating changes in bacterial community

and the toxicity values


Chemical media and inoculated consortium

The fraction of creosote used in this study was composed of 26 of PAH (naphthalene

05 phenanthrene 51 anthracene 122 pyrene 31 dibenzofurane 13 and

acenaphthene47) Creosote was diluted in acetonitrile (purchased form Sigma Aldrich

Steinheim Germany) in a stock solution of a final concentration of 0439 gmiddotml-1 containing

0117 g PAHmiddotml-1 The culture mediums LuriandashBertani (LB) and Bushnell-Haas Broth (BHB)

were purchased from Panreac (Barcelona Spain) Biostimulated treatments were amended

with BHB as inorganic nutrients source which composition was optimized for PAH-degrading

consortium (C2PL05 consortium) in a previously work (Simarro et al 2010) with the optimum

composition 02 gmiddotlminus1 MgSO4 7H2O 002 gmiddotlminus1 CaCl2 2H2O 0281 gmiddotlminus1 KHPO4 0281 gmiddotlminus1

K2HPO4 002 gmiddotlminus1 NH4NO3 0195 gmiddotlminus1 Fe2(SO4)3 According to Bautista et al(2009) Tween-

80 was the optimal surfactant for PAH biodegradation by bacteria and was added in a critical

micellar concentration (CMC) of 300 μlmiddotml-1 (0012 mM) Bioaugmented treatments were

inoculated with the PAH-degrading consortium C2PL05 extracted from a permanently PAH

contaminated soil from a refinery in Ciudad Real (Spain) and previously identified and

described in Molina et al(2009)

Experimental design

Five different treatments in microcosms designated as T1 to T5 (see Table 1) were carried

out each in duplicate for five sampling times zero 6 40 145 and 176 days from December

2009 to May 2010 In total 40 microcosms containing 550g of natural soil samples collected

from an uncontaminated area of Rey Juan Carlos University in Madrid (Spain) were carried

out The soil obtained from the first top 20 cm and sieved by 2 mm was located in plastic


149

trays and randomly arranged outdoor in terrace and protected with a plastic to avoid the rain

and snow on them Each tray except the treatment T1 contained 56 ml of a creosote

solution in n-hexano (0439 g ml-1) with final amount of creosote per tray of 25 g

Microcosms were maintained at 40 of water holding capacity (WHC) considered as

optimum during bioremediation process (Vintildeas et al 2005) For biostimulation microcosms

samples were hydrated with the required amount of the optimum BHB while in treatment no

biostimulated samples were hydrated with only mili-Q water Bioaugmented treatments were

inoculated with 5ml of the consortium C2PL05 (2015x107 plusmn 427x106 cellsmiddotg soil-1 of

heterotrophic microorganisms and 177x105 plusmn 101x105 cellsmiddotg soil-1 of creosote-degrading

microorganisms)

Table 1 Summary of the treatment conditions

Code Treatments Conditions

T1 Untreated soil (control) Uncontaminated soil

T2 Natural attenuation Contaminated soil with 56 ml creosote moistened 40WHC

with 1054 ml mili-Q water

T3 Biostimulation Contaminated soil with 56 ml of creosote stock solution

moistened 40WHC with 1104 ml BHB

T4 Bioaugmentation Contaminated soil with 56 ml of creosote stock solution

moistened 40WHC with 1054 ml mili-Q water 5 ml consortium

C2PL05

T5 Biostimulation

+ Bioaugmentation

Contaminated soil with 56 ml of creosote stock solution

moistened 40WHC with 1054 ml BHB inoculated with 5 ml

Characterization of soil and environmental conditions

The water holding capacity (WHC) was measured following the method described by Wilke

(2005) and the water content was calculated through the difference between the wet and dry

weigh after drying at 60ordmC during 1 hour pH was measured using a GLP 21 micro pHmeter

(Crison Barceona Spain) resuspending 1 g of the soil in mili-Q water (110) and incubating it

in an orbital shaker at 150 rpm at 25 ordmC during 1 h Temperature which the experiments were

developed was recorded on a temperature loggers (Tidbit Loggers Onset Computer

Pocasset Mass) located in the site

Total heterotrophic microorganisms (HM) and creosote-degrading microorganisms

(C-DM) of the microbial population of the natural soil was counted using a miniaturized most

probable number technique (MPN) in 96-well microtiter plates with eight replicates per

dilution (Wrenn amp Venosa 1996) The number of cells was calculated with Most Probable


150

Number Calculator software Version 404 (Klee 1993)To extract the microorganisms from

the soil 1 g of soil was resuspended in 10 ml of phosphate buffer saline (PBS) and was

shaker at 150 rpm at 25 ordmC during 24 h The HM were determined in 180 μl of LB medium

with glucose (15 gl-1) and C-DM were counted in 180 μl of BHB medium with 10 μl of

creosote stock solution as carbon source

Respiration and toxicity assays

To measure the respiration during the experiments 10 g of soil moistened with 232 ml of

mili-Q water (to maintain a water capacity of 40 WHC) was incubated in duplicate in a

desiccator during 14 days at 25ordmC Replicates contained 14 ml of NaOH 02 M to absorb the

CO2 produced by microorganisms The vials were periodically replaced and checked

calorimetrically with HCl (01M) and phenolphthalein as indicator The test was doing with

BaCl2 (01 M) in excess to ensure the precipitation of carbonates The numbers of moles of

CO2 produced were calculated as a difference between initial moles of NaOH in the

replicates and moles of NaOH checked with HCl (moles of NaOH free)

The toxicity evolution during the PAH degradation was also monitored through a short

screening of the samples with the Microtox TM method with the luminescent bacterium Vibrio

fischeri following the protocol suggested by Microbics Corporation (1992) The toxicity was

expressed as the percentage of the V fischeri inhibition after 15 min of incubation at 15ordmC

Monitoring the removal of creosote and polycyclic aromatic hydrocarbons

Organic compounds were extracted and analyzed from the microcosms samples at 0 6 40

145 and 176 days by gas chromatography-mass spectrometry (GC-MS) to estimate the

creosote and percentage of PAH depletion A gas cromatograph (model CP3800 Varian

Palo Alto CA USA) equipped with a Varian Factor Four VF-1ms capillary column (15 m

length 025 mm ID 025 μm film thickness) was coupled to a quadruple mass-spectrometer

detector (Model 1200L Varian) The stationary phase was composed by phenyl (5) and

dimethylpolysiloxane (95) as carried gas ice in the mobile phase Temperature gradient

program used was initial temperature of 80 ordmC for 2 min temperature increase to 300 ordmC at

the rate of 1ordmC min-1 final temperature of 300 ordmC for 15 min with a final duration of the

method of 39 min Organic compounds were extracted with 100 ml of dichloromethane


residue was dissolved in 1 ml of dichloromethane an encapsulated in a vial to inject 05 μl in


151

the FDI chromatograph The concentration of each PAH and creosote was calculated from

the chromatograph of the standard curves

DNA extraction molecular and phylogenetic analysis for characterization of the total

microbial population in the microcosms

Non culture-dependent molecular techniques as denaturing gradient gel electrophoresis

(DGGE) was performed to identify non-culture microorganisms and to compared the

biodiversity between treatments and its evolution at 145 and 176 days of the process Total

community DNA was extracted from 25 g of the soil samples using Microbial Power Soil

DNA isolation kit (MoBio Laboratories Solano Beach CA USA) In total suitable yields of

high molecular-weight DNA (5-20 μgg of soil-1) were obtained The V3 to V5 variable regions

of the 16S rRNA gene were amplified using the primers set 16S 518R and 16S 338F-GC


Primer 338F-GC included a GC clamp at the 5acuteend (5acute-CGC CCG CCGCGC CCC GCG

CCC GTC CCG CCG CCC CCG CCC G-3acute) 20 μl of PCR product was loaded on to a 10

(wtvol) polyacrylamide gels that was 075mm tick and the denaturing gradients used ranged

from 35 to 65 denaturant (more details in Gonzalez et al 2011) Gel were stained with

Syber-Gold and viewed under UV light and predominant bands were excised and diluted in

50μl of mili-Q water Due to impossibility of reamplified bands DNA of the bands was cloned

in the pGEM-T Easy Vector (Promega Madison WI) Plasmids were purified using the High

Pure plasmid Isolation Kit (Roche) and sequenced using the internal primers 338F and 518R

Creosote-degrader uncultured bacterium (DUB) were edited and assembled using version

487 of the BioEdit program (Hall 1999) BLAST search (Madden et al 1996) was used to

find nearly identical sequences for the 16S rRNA sequences determined All DUB identified

sequence and 25 similar sequences downloaded from GenBank were used to perform the

phylogenetic tree Sequences were aligned using the Q-INS-i algorithm (Katoh amp Toh 2008a)

of the multiple sequence alignment software MAFFT version 6611 (Katoh amp Toh 2008b)

aligning sequences in a single step Sequence divergence was computed in terms of the

number of nucleotide differences per site between of sequences according to the Jukes and

Cantor algorithm (1969) The distance matrix for all pair wise sequence combinations was

analyzed with the neighbour-joining method (NJ) of phylogenetic tree construction with 1000

bootstrap replicates by using version PAUP 40B10 (Swofford 2003) In addition maximum

parsimony (MP) was also analyzed (Molina et al 2009) Sequences of Sphirochatea

americans belonged to Sphirochaetes phylum were used as out-group (Gupta amp Griffiths

2002) Images of DGGE were digitalized and DGGE bands were processed using the UN-

Scan-It gel analysis software version 60 (Silk Scientific US)


152


In order to evaluate the effects of treatments on intrinsic growth rate (μ) toxicity degradation

of organic compounds and respiration analysis of variance (ANOVA) were used The

variances were checked for homogeneity by the Cochranacutes test Student-Newman-Keuls

(SNK) test was used to discriminate among different treatments after significant F-test

representing these differences by letters in the graphs Data were considered significant

when p-value was lt 005 All tests were done with the software Statistica 60 for Windows

Differences in microbial assemblages by biostimulation by bioaugmentation and by time

(145 and 176 days) were graphically evaluated with a non-metric multidimensional scaling

(MDS) using PRIMER software (Clarke 1993) The previous period to 145 days was

considered a period of cold conditions and the time from 145 to 176 days a period of higher

temperatures SIMPER method was used to identify the percent contribution of each band to

the similarity in microbial assemblages between factors Bands were considered ldquohighly

influentialrdquo if they contributed to the first 60 of cumulative percentage of average similarity

betweenwithin combination of factors In addition Shannon index (Hacute) was calculated from

DGGE bands applying equation 2 to estimate the ecological diversity of each treatment at

136 and 145 days

Equation 2

where pi is the proportion in the gel of the band i with respect to the total of all bands

detected calculated as coefficient between band intensity and total intensity of all

bands (Baek et al 2007)

Results

Physical chemical and biological characteristics of the natural soil used for the treatments

pH of the soil was slightly basic 84 and the water content of the soil was 10 although the

soil had a high WHC (521) possibly due to their sandy character Initial proportion of C-DM

from natural soil represented only 088 of the total heterotrophic population with a number

of microorganisms two order of magnitude higher (201 x 107 plusmn 427 x 106 cells g soil-1)

Figure 1 shows that the evolution of the monthly average temperature observed during the

experiment and the last 30 years Average temperature decreased progressively from

October to January from 16 ordmC to a minimum average of 6 ordmC starting to increase

progressively to reach a mean value of 21 ordmC in June


153

October

November

DecemberJanuary

FebruaryMarch

April MayJune

468

10121416182022

0 day

40 day

145 day

176 day

6 dayT

empe

ratu

re (

ordmC)

Month

Figure 1 evolution of the normal values of temperature (square) and evolution of

the monthly average temperature observed (circle) during the experiment

Respiration of the microbial population

Table 2A shows the ANOVA results concerning to the accumulated values of CO2 produced

for each treatment in each time period (from 0- to 6 days from 40 to 145 days and from 145

to 176 days) Due to interval time was the only significant factor (Table 2A) differences in

percentage of accumulative CO2 by sampling times (6 40 145 and 176 days) were analyzed

and showed in Figure 2 Differences between sampling times showed that the accumulated

percentage of CO2 was significantly higher at 176 days than at other time

6 40 145 17600

10x10-4

20x10-4

30x10-4

40x10-4

50x10-4

a a

b

aCO

2 mol

esg

of

soil

Time (days)

Figure 2 accumulated CO2 issue at 6 40 145 and 176 days Error bars show the

standard deviation and the letters show significant differences between groups

(plt005 SNK)


154

Toxicity assays

Changes on the toxicity during the creosote degradation process (Figure 3A) showed that all

treatments had a similar evolution T1 (uncontaminated) was not toxic (lt20 ) but toxicity of

treatments with creosote increased constantly from initial value of 26 to a values higher

than 50 Only during last period of time (145 to 176 days) toxicity started to decrease

slightly Despite similar toxicity values reached with the treatments interaction between time

periods (0 to 6 days 40 to 145 days and 145 to 176 days) and treatments showed significant

differences (Table 2B) Differences between groups by both significant factors (Figure 3B)

showed that toxicity of all treatments in first time period was significantly lower than in the

other periods Differences in toxicity between the two last periods were only significant for

treatment T4 in which toxicity increase progressively from the beginning

0 6 20 40 56 77 84 91 98 1051121251321411760

10

20

30

40

50

60

70

80

90

100 BA

Tox

icity

(

)

Time (days)T2 T3 T4 T5

c

c

c

b

c

bc

bcbc

aa

aa

Treatment

Figure 3 (A) evolution of the toxicity () of the treatment T1 (square) T2 (circle) T3 (triangle) T4

(inverted triangle) and T5 (rhombus) during the experiment (B) percentage of toxicity of the treatment

in the interval times 1 (black bars) from 0- to 6 days interval 2 (with bars) from 6 to 40 days and

interval 3 (gray bars) from 40 to 176 days Error bars show the standard deviation and letters



155

Biodegradation of creosote and polycyclic aromatic hydrocarbons

The results concerning the chromatography performed on the microcosms at 0 40 145 and

176 days are shown in Figure 4 Creosote depletion during first 40 days was very low

compared with the intensive degradation occurred from 40 to 145 days in which the greatest

amount of creosote was eliminated (asymp 60-80) In addition difference between residual

concentration of PAH at final time by type of PAH (phenanthrene anthracene and pyrene)

and treatment were analyzed (Table 2C) Both factor were significantly influential although

was not the interaction between them Differences by PAH (Figure 4B) showed that

anthracene degradation was significantly higher than other PAH and differences by

treatments (Figure 4C) showed that difference were only significant between treatment T3

and T2 lower in the treatment T3


156

T1 T2 T3 T4 T50000

0005

0010

0015

0020

0025

0030

0035

0040

g cr

eoso

te

g so

il

Phenanthrene Anthracene Pyrene0

102030405060708090

100

C

aab

abb

a

bb

B

A

Ave

rage

res

idua

l con

cenr

atio

n of

PA

H (

)

T2 T3 T4 T50

102030405060708090

100

Tot

al r

esid

ual c

once

ntra

tion

of

PA

H (

)

Figure 4 (A) creosote depletion during the experiment at sampling times 6 days (black

bars) 40 days (with bars) 145 days (grey bars) and 176 days ( ) (B) average residual

concentration at 176 days of the identified PAH (phenanthrene anthracene and pyrene)

and (B) average residual concentration of the identified PAH as a function of applied

treatment (C) Error bars show the standard error and the letters show significant

differences between groups (plt005 SNK)


157

Table 2 Analysis of variance (ANOVA) of the effects on the μ of the

heteroptrophic population (A) μ of the creosote degrading microorganisms (B)

accumulated value of CO2 (C) toxicity (D) and residual concentration of PAH (E) SS is

the sum of squares and df the degree of freedoms

Factor df SS F P

C) Accumulated value of CO2 (n=40) Time interval 3 65-5 3112

Treatment 4 60-6 202 ns

Interval x Treatment 12 11-5 134 ns

Error 20 14-5

D)Toxicity (n=24) Time interval 2 907133 11075

Treatment 3 12090 098 ns

Interval x Treatment 6 122138 497

Error 12 49143

E) Residual concentration of the PAH (n=24) Treatment 3 95148 548

PAH 2 168113 1452


Error 12 69486

p-value lt 005

p-value lt 001

p-value lt 0001

Diversity and evolution of the uncultivated bacteria and dynamics during the PAH

degradation

The effects of different treatments on the structure and dynamics of the bacterial community

at 145 days and 176 days were analyzed by DGGE (Figure 5) At 145 days 8 bands (3 4 10

810 16 17 26 and 27 see Figure 5) were cloned and 6 different genotypes (DUB 12-RS to

DUB-17RS) were identified whereas at 176 days 5 bands (4 112 113 22 and 26 see

Figure 5) and 6 different genotypes (DUB-12RS DUB-13RS DUB-18RS DUB-19RS DUB-

20RS and DUB-21RS) were identified Most influential bands considered as 60 of

contribution to similarity according to the results of PRIMER analysis is showed at the Table

3 Similarities between treatments at 145 and 176 days were compared and analyzed as a

function of the addition of nutrients (biostimulated vs no biostimulated treatments) and the

addition of degrading consortium C2PL05 (bioaugmentated vs no bioaugmentated

treatments) The addition of nutrients was the factor that best explained differences between

treatments and so results in Table 3 are as a function of the addition of nutrients At 145

days no-biostimulated treatments T1 T2 and T4 were only similar in 402 whereas


158

biostimulated treatments (T3 and T5) were 6567 (Table 3) The patter were slightly

opposite at 176 days where no-biostimulated treatments were more similar (7026 ) than

biostimulated treatments (average similarity 4384 ) In addition at 145 days (Figure 6A)

natural attenuation (T2) was the only similar treatment to microbial community from the

uncontaminated treatment (T1) However at 176 days (Figure 6B) bacterial communities

from all treatments were highly different to the treatment T1 and there was no defined group

In addition PRIMER analysis allowed us to estimate the Shannon diversity index (Hacute) for

each treatments at 145 and 176 days indicating that the bacterial diversity increased for the

treatments T1 (3328) T3 (4154) and T5 (3739) remarkably higher in the treatment T4

Table 3 Bands contribution to 60 similarity primer between treatments grouped by

treatments biostimulated and no biostimulated at 145 days and 176 days Average

similarity of the groups determined by SIMPER method

145 days

Band DUB No biostimulated (T1 T2 T4) Biostimulated (T3 T5)

3 DUB-12RS

DUB-17RS 2875

16 DUB-17RS 1826

17 DUB-12RS

DUB-16RS 1414

18 Unidentified 3363


Cumulative similarity () 6725 6115 Average similarity () 402 6567

176 days


11 Unidentified 2116 13 Unidentified 2078 1794

23 Unidentified 2225 2294

26 DUB-13RS 1296


bands from DGGE unidentified after several attempts to clone


159

Figure 5 Denaturing gradient gel electrophoresis (DGGE) at 145 days (A) and 176 days (B) of PCR-

amplified 16S rDNA gen fragments from the consortium C2PL05 (lane B) control experiment (lane C)

treatment with natural attenuation T1 (lane T1)biostimulated treatment T2 (lane T2) bioaugmentated

treatment T3 (lane T3) bioaugmentated treatment T4 (lane T4) and biostimulated and

bioaugmentated treatment t5 (lane T5) Lane A is the molecular weigh marker Numbers are the

bands cloning


160

Figure 6 proximity analysis (MDS) based on the Bray-Curtis dissimilarity

matrix of each treatment from the bands obtained in DGGE at 145 days (A)

and 176 days (B)


Phylogenetic relationships of the degrading uncultured bacteria are shown in Figure 7 The

aligned matrix contained 1373 unambiguous nucleotide position characters with 496

parsimony-informative Parsimony analysis of the data matrix yielded 87 parsimonious trees

with CI = 0671 RI = 0767 and a length of 1452 Figure 6 also shows the topology of the

maximum parsimony (MP) tree with the bootstrap values of the maximum parsimony and

neighbour joining analyses Inconsistencies were not found between parsimony and

neighbour joining (NJ) topology

Phylogenetic tree was composed by bacteria belonged to Proteobacteria (α- and γ-

Proteobacteria) and Bacteroidetes phylum From DUB-12RS to DUB-17RS were located in

the Pseudomonadaceae clade in which it can be observed five clearly species groups DUB-

13RS and DUB-15RS identified as Pseudomonas trivialensis (HM134251) and P poae

(HM640290) respectively were in an undifferentiated group supported by P trivialensis and

P poae type-strains DUB-14RS similar to P viridiflava (HM190224) formed a group

supported by P viridiflavaT (HM190229) DUB-12RS 98 similar to P fluorescens (GQ


161

496662) was grouped with P fluorescensT (D84013) DUB-16RS was identified as

uncultured Pseudomonas sp (HQ677222) and classified in an indefinite group Finally the

last group of the Pseudomonadaceae clade was formed by DUB17-RS 98 similar to P

parafulva (HQ406758) and grouped with P parafulvaT (D84015) DUB-21RS was nested in

the Enterobacteriaceae clade due to it was identified with 99 of similarity as Pantoea

Brenneri (HM163514) This clade is supported by types-strains of other species of Pantoea

as Pantoea agglomeransT (FJ613819) and other enteric bacteria as Enterobacter cloacaeT

(AJ251469) DUB nested in Enterobacteriaceae and Pseudomonadaceae clade were γ-

Proteobacteria In α-Proteobacteria class are included Rhizobiales and

Sphingomonadaceae clades In the first clade formed by uncultured Balneimonas and

Rihzobiales bacterium supported by Balneimonas floculansT was nested DUB-19-RS 99

similar to an uncultured Balneimonas strain (HM799006) In Sphingomonadaceae clade was

nested DUB-20RS identified as uncultured Sphingomonadales bacterium DUB-18RS was

similar in 99 to Uncultured Flexibacteriaceae bacterium and was nested in Cytophagaceae

clade belonging to Bacteroidetes phylum


162


degrading uncultured bacteria (DUB) obtained from DGGE of the treatments 145 and 176 days of the

process Boostrap values of neighbourjoining and parsimony higher than 50 are showed on the

branch of the tree (NJMP) No incongruence between parsimony and neighbour joining topology were

detected Pseudomonas genus has been designated as P Pantoea genus as Pa Balneimonas as B

and Bacteriovorax as Ba Hidrogenymonas as H Flexibacerium as F T= type strain


163

Discussion

The estimated time of experimentation (176 days) was considered adequate to the complete

bioremediation of the soil according to previous studies developed at low temperatures (15

ordmC ndash 5 ordmC) in which toxicity was reduced below 20 in 101 days and PAH were removed in

137 days above 60 (Simarro et al under review) However our results confirm that

toxicity evaluation of the samples is necessary to know the real status of the polluted soil

because despite creosote was degraded almost entirely (Figure 4A) at the end of the

experiment toxicity remained constant and high during the process (Figure 3A) Possibly the

low temperatures under which was developed the most of the experiment slowed the

biodegradation rates of creosote and its immediate products which may be the cause of

such toxicity In addtion the most removal of creosote (Figure 4A) and higher respiration

rates (Figure 2) occurred from 40 days when temperature began to increase Hence our

results according to other authors (Margesin et al 2002) show that biodegradation at low

temperatures is possible although with low biodegradation rates due to slowdown on the

diffusion rate PAH bioavailability and metabolisms rates (Haritash amp Kaushik 2009 Leahy amp

Colwell 1990)

As in a previously work (Margesin amp Schinner 2001) no significant differences were

observed between treatments in degradation of creosote The final percentage of creosote

depletion above 60 in all treatments including natural attenuation confirm that indigenous

community of the soil degrade creosote efficiently Concurring with these results high

number of creosote-degradaing microorganisms were enumerated in the natural soil at the

time in which the disturbance occurred There is much controversy over whether

preexposure to a pollutant is required for degradation (Johnsen amp Karlson 2005) or if it is a

characteristic intrinsically present in some species of the microbial community that is

expressed when community is exposed to a pollutant (Tian et al 2008 Spain amp van Veld

1983) According to Tian et al 2008) and similarly as in previuosly work in which a wood

degrading consotium from a free polluted soil degraded PAH efficiently bacterial consortium

from natural soil never preexposed to creosota was able to efficiently degrade the

contaminant

Traditionally is widely tested (Yachi amp Loreau 1999) and accepted that higher

diversity leads to greater protection against disturbances (Vilaacute 1998) because the

functionality is higher Bacterial diversity of the biostimulated treatments (T3 and T5) notably

increased during the biodegradation process and showed (T3) a significantly enhance of the

PAH depletion Hence the higher biodiversity of the biostimulated treatment could contribute


164

to the increased of PAH degradation Overall the soil microbial community was significantly

altered in the soil with the addition of creosote is evidenced by the reduction of the size or

diversity of the various population of the treatments precisely in treatments no biostimulated

Long-term exposure (175 days) of the soil community to a constant stress such as creosote

contamination could permanently change the community structure as it observed in DGGEN

AND mds Bioaugmentation (T4 and T5) not resulted in a significant increase of the reduction

of creosote or PAH possibly due to the high adaptability of the indigenous consortium to

degrade PAH The relationship between inoculated and autochthonous consortium largely

condition the results of bioaugmentation Some authors (ie Herwijnen et al2005 Andrenoi

amp Gianfreda 2007) purpose that this technique only has positive effects when indigenous

consortium is no capable to degrade The indigenous microbial community demonstrated

capacity to degrade creosote explains the ineffectiveness of bioaugmentation A study of the

bacterial communities during a bioremediation process is important because such as

demonstrate our results bioremediation techniques cause changes in microbial communities

Most of the DUB identified have been previously related with biodegradation process

of PAH creosote andor diesel 60 of the DUB identified (DUB-12RS to DUB17RS)

belonged to Pseudomonas genus widely studied in bioremediation (ie Ma et al 2006

Molina et al 2009) Our results showed that it was the unique representative group at 145

days and the most representative at 176 days of the biodegradation process However in

this work it has been identified some species of Pseudomonas grouped in P trivialis P poae

and P Viridiflava clades (DUB-13RS DUB-15RS and DUB-14RS respectively) less

commonly described in biodegradation process (ie Bogan et al 2003) α-Proteobacteria

class was composed by DUB-19RS (Uncultured balneimonas) and DUB-20RS (Uncultured

Sphingomonadales bacterium) DUB-18RS belonged to phylum Bacteroidetes previously

identified in degradation of high-molecular-mass organic matter in marine ecosystems in

petroleum degradation process at low temperatures and in PAH degradation during

bioremediation of creoste-contaminated soils (Cotrell amp Kiechman 2000a Brakstad et al

2006 Vintildeas et al 2005) Something important to emphasize is the identification of the

Pantoea brenneri (DUB-21RS Enterobacteriaceae clade) and an uncultured Balneimonas

bacteria (DUB-19RS Metylbacteriaceae Rhizobiales clade) as creosote degrader because

have not been previously described as such However very few reports have indicated the

ability to degrade PAH of some genera of the enteric bacteria group as Enterobacter (Molina

et al 2009) Klebsiella (Grant et al 1967) or Escherichia (Diaz et al 2001)

In conclusion temperature is a very influential factor in ex situ biodegradation process

that control biodegradation rates toxicity reduction availability of contaminant and bacterial

metabolisms and so is an important factor to take into account during bioremediation


165

process Biostimulation was the technique which more efficiently removed PAH compared

with natural attenuation In this work bioaugmentation not resulted in an increment of the

creosote depletion probably due to the ability of the indigenous consortium to degrade

Bioremediation techniques produce change in the bacterial communities which is important

to study to evaluate damage in the habitat and restore capability of the ecosystem


166

References

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habitats Appl Microbiol Biotechnol 76 287-308

Atagana HI 2006 Biodegradation of polycyclic aromatic hydrocarbons in contaminated soil

by biostimulation and bioaugmentation in the presence of copper (II) iron World J

Microbiol Biotechnol 22 1145-1153

Baek SH Kim KH Yin CR Jeon CO Im WT Kim KK amp Lee ST 2003 Isolation and

characterization of bacteria capable of degrading phenol and reducing nitrate under

low-oxygen conditions Curr Microbiol 47462-466




Behrendt U Ulrich A amp Schumann P 2003 Fluorescent pseudomonas associated with the

phyllosphere of grasses Pseudomonas trivialis sp nov Pseudomonas poae sp nov

and Pseudomonas congelans sp nov Int J Syst Evol Microbiol 53 1461ndash1469


at low temperatures (0-5 ordmC) and bacterial communities associated with degradation


Bodour AA Wang JM Brusseau ML amp Maier RM 2003 Temporal changes in culturable

phenatrhene degraders in response to long-term exposure to phenantrhene in a soil

column system Environ Microbiol 5 888-895

Bogan BW Lahner LMamp Sullivan WR 2003 Degradation of straight-chain aliphatic and

high molecular weight polycyclic aromatic hydrocarbons by a strain of Mycobacterium

austroafricanum J Appl Microbiol 94 230-239

Chen J Wong MH amp Tam N 2008 Multi-factors on biodegradation kinetics of plyciclic

aromatic hydrocarbons (PAH) by Sphingomonas sp a bacterial strain isolated from

mangrove sediment Marine Pollut Bull 57 695-702


Austral Ecol 18 117-143

Cotrell MT amp Kirchman DL 2000 Natural assemblages of marine proteobacteria and

members of Cytophaga-Flavobacter cluster consuming low- and high molecular

weight dissolved organic matter Appl Environ Microbiol 66 1692-1697

Couling NR Towel MG Semple KT 2010 Biodegradation of PAH in soil Influence of

chemical structure concentration and multiple amendment Environ Pollut 158

3411-3420


167

Diaz E Fernandez A Prieto MA amp Garcia JL 2001 Bioremediation of aromatic

compounds by Eschericlia coli Microbiol Mol Biol Rev 65 523-569

Dowty RA Shaffer GP Hester MW Childers GW Campo FM amp Greence MC 2001

Phytoremediation of small-scale oil spills in fresh marsh environments a mesocosm

simulation Marine Environ Res 52 195-211



the bacterial community during the process Bioresource Technol 102 9438ndash9446

Grant DJW 1967 Kinetic aspect of the growth of Klebsiella aerogenes with some

benzenoid carbon sources J Gen Microbiol 46 213-224

Hall TA 1999 bioedit a user-friendly biological sequence alignment editor and analysis

program for Windows 9598NT Nucleic Acids Symp Ser 4195-98

Haritash AK Kaushik CP 2009 Biodegradation aspects of Polycyclic Aromatic

Hidrocarbons (PAH) A review J Hazard Mater 169 1-15

Herwijnen R Joffe B Ryngaert A Hausner M Springael D Govers HAJWuertz S amp

Parson JR 2005 Effect of bioaugmentation and supplementary carbon sources on

egradation of polycyclic aromatic hydrocarbons by a soil-derived culture FEMS

Microbiol Ecol 55 122-135

Johnsen A amp Karlson U 2005 PAH degradation capacity of soil microbial communities-does

it depend on PAH exposure Microbial Ecol 50 488ndash495

Jukes TH amp Cantor CR 1969 Evolution of protein molecules Pp 21-132 in Munro HN ed

Mammalian protein metabolism Academic Press New York

Kaplan CW Kitts CK 2004 Bacterial succession in a petroleum land treatment unit Appl


Karen M amp Chistoserdov AY 2001 Phylogenetic analysis of the sucession of bacterial

communities in the Great South Bay (Long Island) Microb Ecol 35 85-95





Klee AJ 1993 A computer program for the determination of the most probable number and

its confidence limits J Microbiol Methods 18 91-98

Leahy JG amp Colwell RR 1990 Microbial degradation of hydrocarbons in the environment

Microbiol Mol Biol R 54 305-315

Loacutepez Z Vila J Ortega-Calvo JJ amp Grifoll M 2008 Simultaneous biodegradation of

creosote-polycyclic aromatic hydrocarbons by a pyrene-degrading Mycobacterium

Appl Microbiol Biotechnol 78 165-172


168

MaY Wang L amp Shao Z 2006 Pseudomonas the dominant polycyclic aromatic

hydrocarbon-degrading bacteria isolated from Antarctic soils and the role of large

plasmids in horizontal gene transfer Environ Microbiol 8 455ndash465

Madden TL Tatusov RL amp Zhang J 1996 Applications of network BLAST server Methods




3127-3133



McConkey BJ Duxbury CL Dixon DG amp Greenberg BM 1997 Toxicity of a PAH

photooxidation product to the bacteria Photobacterium phosphoreum and the

duckweed Lemna gibba Effects of phenanthrene and its primary photoproduct

phenanthrenequinone Environ Toxicol Chem 16 892-899


Microbial population changes during bioremediation of an experimental oil spill App


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Mills MA Bonner JS Page CA amp Autenrieth RL 2004 Evaluation of bioremediation

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Kuhad RC Ward OP (eds) Adv Appl Biorem p103-121 Springer Berliacuten





15


biodegradation of polycyclic aromatic hydrocarbons in mangrove sediments of

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169


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Vilagrave M 1998 Efectos de la diversidad de especies en el funcionamiento de los ecosistemas

Orsis 13 105-117









42 252-258

Yachi S amp Loreau M 1999 Biodiversity and ecosystem productivity in a fluctuating

environment The insurance hypothesis Proc Natl Acad Sci USA 96 1463-1468

Yu SH Ke L Wong YS amp Tam NFY 2005a Biodegradation of polycyclic aromatic

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Yu KSH Wong AHY Yau KWY Wong YS amp Tam NFY 2005b Natural attenuation

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bull Discusioacutengeneral

II

Discusioacuten general

173


Temperatura y otros factores ambientales determinantes en un proceso de

biodegradacioacuten

El resultado final de la aplicacioacuten directa de teacutecnicas de biorremediacioacuten en un medio

contaminado estaacute fuertemente influenciado por los paraacutemetros ambientales Por este motivo

son numerosos los estudios (ej Chaicircneau et al 2005 Cheung amp Kinkle 2005 Leys et al

2005 Chen et al 2008) realizados para optimizar y en la medida de lo posible modificar

tanto los factores bioacuteticos como abioacuteticos Frecuentemente la optimizacioacuten se ha llevado a

cabo considerando exclusivamente un solo factor implicado por ejemplo la temperatura

(Torres et al 2005) la concentracioacuten de nutrientes y la relacioacuten CNP (Leys et al 2005) o

el tipo de nutrientes y los surfactantes adicionados (Cheung amp Kinkle 2005) Pocos son los

estudios en los que se valoran dos o tres factores conjuntamente (Kaumlstner et al 1998

Cheung amp Kinkle 2005) y menos auacuten en los que se combinan maacutes de cuatro paraacutemetros

variables Chen et al (2008) destacoacute la importancia de evaluar en el proceso de

optimizacioacuten tanto los efectos individuales como los derivados de la interaccioacuten entre

factores mediante un meacutetodo factorial completo La optimizacioacuten de un proceso de

biorremediacioacuten previo a la aplicacioacuten in situ es fundamental y un disentildeo ortogonal del

experimento va a permitir ademaacutes considerar los efectos sineacutergicos y antagoacutenicos

derivados de la interaccioacuten entre las variables bioacuteticas y abioacuteticas (Chen et al 2008) Los

resultados obtenidos en los dos trabajos de optimizacioacuten que componen el capiacutetulo 1

demuestran que los factores ambientales significativamente influyentes en la tasa de

biodegradacioacuten (kB) de HAP son diferentes cuando se fijan como constantes todos los

paraacutemetros excepto uno (capiacutetulo 1a) que cuando todos los paraacutemetros se consideran

variables y se combinan en un anaacutelisis ortogonal (capiacutetulo 1b) Sin embargo los resultados

obtenidos de un anaacutelisis factorial no invalidan la optimizacioacuten individual la cual es necesaria

y maacutes adecuada en casos en los que tan soacutelo interesa estudiar la influencia de un

determinado factor en el proceso de biodegradacioacuten En algunos casos determinados

paraacutemetros ambientales fijos e inamovibles (ej bajas temperaturas) dificultan el proceso de

biodegradacioacuten y mediante la optimizacioacuten individual y posterior modificacioacuten de otros

factores del medio (ej nutrientes fuente de carbono) se consigue aumentar la eficacia del

proceso Ademaacutes como resultado de la optimizacioacuten indiviadual de factores realizada en el

capiacutetulo 1a de la presente tesis se obtuvo un medio de crecimiento oacuteptimo (capiacutetulo 1) que

que se usoacute en ensayos y experimentos posteriores (capiacutetulos 3 y 4)


174

Dada la elevada variabilidad de resultados que se pueden obtener en la eficacia de

biodegradacioacuten en funcioacuten de los factores ambientales y sus modificaciones proponemos

que los ensayos de optimizacioacuten del proceso para determinar las condiciones oacuteptimas del

mismo es fundamental para maximizar la eficacia (tiempo y costes) de la aplicacioacuten in situ

De entre todos los factores ambientales limitantes de la biodegradacioacuten de

hidrocarburos del petroacuteleo como son los HAP la temperatura es uno de los que maacutes

condiciona la eficacia del mismo (Chen et al 2008) En la presente tesis los procesos de

biodegradacioacuten realizados a bajas temperaturas (capiacutetulo 3 y 4) con el objetivo de evaluar la

influencia de este factor en la eficacia de degradacioacuten y en la comunidad bacteriana

muestran que la degradacioacuten del contaminante es menor a temperaturas inferiores a 15ordmC

(Margesin et al 2002) La notable ralentizacioacuten de las tasas de crecimiento bacteriano y

degradacioacuten a temperaturas bajas (capiacutetulos 3 y 4) fue debida a la menor solubilidad de los

HAP (Haritash amp Kaushik 2009) y al decrecimiento del metabolismo microbiano (Leahy amp

Colwell 1990) Los estudios centrados en la obtencioacuten de la temperatura oacuteptima durante los

procesos de degradacioacuten de HAP por un consorcio bacteriano (Capiacutetulos 1a 1b y 3) han

determinado que no existe un valor oacuteptimo sino maacutes bien un rango oacuteptimo que oscila entre

los 20ordmC y los 30ordmC Tal y como concreta Chen et al (2008) son las fluctuaciones amplias

de temperatura lo que dificulta el proceso de biodegradacioacuten Cuando el rango de variacioacuten

es estrecho y ademaacutes se encuentra dentro de los valores oacuteptimos la influencia es

significativamente despreciable (capiacutetulo 1b) Sin embargo hay que tener presente que

existen ecosistemas susceptibles de ser contaminados en zonas cuyas condiciones

climaacuteticas son extremas Histoacutericamente grandes desastres ecoloacutegicos se han producido en

aacutereas cuyas temperaturas medias estaacuten muy por debajo del rango oacuteptimo como es el caso

del petrolero Exxon Vadez en Alaska (Bence et al 1996) o el Prestige en Espantildea (Soriano

et al 2006) A pesar de la evidente importancia de este aspecto poco se sabe sin embargo

de la degradacioacuten de HAP a temperaturas friacuteas comprendidas entre los 5 ordmC-15 ordmC la cual

es posible gracias a la presencia de especies psicrotolerantes y psicroacutefilas (ldquocold-adaptedrdquo)

(Margesin amp Schinner 2001) Alguna de estas especies que han adquirido o que poseen

intriacutensecamente la capacidad para degradar hidrocarburos pertenecen a los geacuteneros

Pseudomonas Acinetobacter o Pshycrobacter (Eriksson et al 2003 Margesin et al 2003)

La identificacioacuten de estos geacuteneros en los consorcios bacterianos (C2PL05 y BOS08)

posiblemente ha sido determinante en los procesos de biodegradacioacuten a bajas temperaturas

(capiacutetulos 3 y 4) ya que aunque con menores tasas que a temperaturas altas la

biodegradacioacuten ha sido posible Estas especies son claves para el mantenimiento de dicha

comunidad ya que su actividad metaboacutelica durante periodos de bajas temperaturas o en

ambientes permanentemente friacuteos permite la mineralizacioacuten de los hidrocarburos y

subsecuente formacioacuten de otros compuestos maacutes sencillos y faacuteciles de degradar por el resto


175

de las especies del consorcio o la comunidad (Pelz et al 1999) La utilizacioacuten de consorcios

bacterianos adaptados a climas friacuteos compuestos por especies con capacidad degradadora

puede ser una medida de actuacioacuten fundamental en aacutereas extremas Ademaacutes a partir de

estas cepas psicrotolerantes y psicroacutefilas se estaacuten aislando enzimas oxidativas capaces de

trabajar a bajas temperaturas con un importante potencial en procesos biotecnoloacutegicos

(Cavicchioli et al 2002)

Consorcios bacterianos durante un proceso de biodegradacioacuten factores que

determinan la sucesioacuten de especies

La sucesioacuten de especies en un consorcio durante un proceso de biodegradacioacuten depende

en gran medida de ciertas caracteriacutesticas celulares y metaboacutelicas de las especies que lo

componen sobre todo cuando la fuente de carbono son HAP de alto peso molecular

(Mueller et al 1997) Por ejemplo algunas especies de Pseudomonas (P aeruginosa

Soberon-Chavez et al 2005) tienen la capacidad de producir biosurfactantes para aumentar

la biodisponibilidad de HAP o bien como es el caso de las Gram-positivas la presencia de

una uacutenica membrana permite un transporte maacutes eficaz de los HAP al interior de la ceacutelula

(Mueller et al 1997) En la mayoriacutea de los casos las sustancias que se presentan como

recalcitrantes para una especie individual pueden metabolizarse a traveacutes de secuencias

cataboacutelicas complementarias que presentan las diferentes especies de un consorcio

(Fritsche 1985) De ahiacute que con los consorcios microbianos se puedan obtener tasas de

degradacioacuten mucho maacutes elevadas que con cepas individuales (Bautista et al 2009) Sin

embargo la estabilidad y eficacia de un consorcio bacteriano estaacuten determinadas por las

relaciones de supervivencia entre las especies que lo componen Un caso en el que las

asociaciones bacterianas son fundamentales son los procesos de biorremediacioacuten a bajas

temperaturas ya que tal y como Leahy amp Cowell (1990) exponen los consorcios bacterianos

cuentan con una capacidad enzimaacutetica maacutes amplia que especies aisladas y por tanto

mayor versatilidad y superioridad de supervivencia

Una modificacioacuten sobre el consorcio como es la dilucioacuten del mismo (capiacutetulo 1b)

puede afectar a las tasas de degradacioacuten finales precisamente porque modifican las

relaciones inicialmente establecidas entre las especies Igualmente este paraacutemetro puede

modificar el patroacuten de crecimiento sin que esto suponga un cambio significativo en la tasa de

degradacioacuten (capiacutetulo 1b) hecho que dependeraacute de la capacidad degradadora de la especie

favorecida (Szaboacute et al 2007) Por tanto la concentracioacuten del inoacuteculo introducido en un

medio contaminado puede condicionar la eficacia del proceso


176

En los diferentes experimentos que componen los capiacutetulos de este proyecto doctoral

no se han realizado pruebas fisioloacutegicas para estudiar las rutas metaboacutelicas o la importancia

relativa del cometabolismo que condicionen la sucesioacuten o composicioacuten de especies de una

comunidad Sin embargo los anaacutelisis de la comunidad bacteriana a traveacutes de la

identificacioacuten de especies cultivables (capiacutetulos 2 y 3) y no cultivables (capiacutetulos 2 3 y 4)

mediante teacutecnicas moleculares indican que la comunidad bacteriana cambia y por tanto

existe una sucesioacuten y coexistencia determinada de especies en el tiempo Los resultados

obtenidos en el capiacutetulo 2 indican que las especies del consorcio cambian en funcioacuten de la

fuente de carbono disponible (HAP y subproductos de degradacioacuten de HAP) y la presencia

de determinados surfactantes (Tween-80 y HAP o solo HAP) por lo que eacutestos pueden ser

factores que intervienen en la sucesioacuten de especies en un consorcio En muchos procesos

de biodegradacioacuten es comuacuten la adicioacuten de ciertas fuentes de carbono para aumentar la

biomasa de los consorcios bacterianos o cepas concretas y acelerar por tanto el proceso de

biodegradacioacuten (Chen amp Aitken 1999 Lee et al 2003) Sin embargo dada la mencionada

influencia de la fuente de carbono sobre la composicioacuten de especies el resultado de esta

medida puede ser negativo en consorcios bacterianos en los que coexistan especies

degradadoras de una determinada fuente de carbono (ej HAP) con otras que no lo son

(capiacutetulo 1a y 1b) Este hecho es debido a que la adaptacioacuten a la nueva fuente de carbono

de los microorganismos degradadores de HAP se traduce en un aumento de la fase de

latencia y por tanto en un retraso de la tasa de degradacioacuten (Maier et al 2009) Este

fenoacutemeno se pudo observar con claridad cuando se suministroacute al consorcio degradador

C2PL05 glucosa como uacutenica fuente de carbono o en combinacioacuten con HAP (capiacutetulos 1a y

1b)

Nuevas especies bacterianas degradadoras de HAP

La identificacioacuten de especies en los numerosos trabajos de biodegradacioacuten realizados hasta

el momento verifican la existencia de una importante variedad de bacterias degradadoras

de HAP Sin embargo esto no implica que no haya taxa particularmente bien adaptados a

medios contaminados (Mueller et al 1997) y que frecuentemente esteacuten involucrados en

procesos de biodegradacioacuten Este es el caso de geacuteneros como Pseudomonas

Acinetobacter Sphingomonas y Stenothrophomonas identificados durante los ensayos que

componen los capiacutetulos 2 3 y 4 Ademaacutes de la identificacioacuten de especies pertenecientes a

estos geacuteneros ampliamente descritos en procesos de biodegradacioacuten (ej Pseudomonas

Sphingomonas Sphingobium Ralstonia Flexibacter Rhodococcus y Bacillus) cabe

destacar la importancia del aislamiento e identificacioacuten de secuencias englobadas en nuevos

geacuteneros degradadores ineacuteditos hasta el momento o cuya implicacioacuten en estos procesos es


177

escasa Este es el caso de Enterobacter cloacae y E ludwigii (γ-Proteobacterias)

identificadas en el consorcio C2PL05 como degradadoras de HAP de bajo peso molecular

Incluso en un estudio previo (Bautista et al 2009) se ha determinado que la eficacia

degradadora de este geacutenero es mucho maacutes eficaz que otras especies degradadoras

frecuentemente descritas como Pseudomonas fluorescens o Stenotrophomonas maltophilia

Pantoea aglomerans (γ-Proteobacterias) es otra Enterobacteriaceae identificada por primera

vez como degradadora de (capiacutetulo 4) En escasas ocasiones (Toledo et al 2006) una

especie de la familia Enterobacteraceae ha sido identificada como degradadora de HAP o

de crudo (Zhang et al 2010) Asiacute mismo es la primera vez que bacterias Gram-positivas

pertenecientes a los geacuteneros Balneimonas sp (capiacutetulo 4) Bradyrhizobium sp y

Nitrobacteria sp (capiacutetulo 2) todas α-Proteobacterias y por otro lado el geacutenero

Microbacterium sp (Phylum Actinobacterias Capiacutetulo 3) se relacionan con procesos de

biodegradacioacuten de HAP y en procesos de mineralizacioacuten de sustancias recalcitrantes La

presencia de estos organismos debe quedar justificada por su capacidad degradadora dado

que han sido identificadas a partir de bandas de gran intensidad en DGGE teacutecnica que se

ha realizado con muestras procedentes de ensayos de biodegradacioacuten de HAP y creosota

(capiacutetulos 2 3 y 4) Es decir su mera presencia en el consorcio no parece justificable por

causas aleatorias sino maacutes bien por su implicacioacuten directa en los procesos metaboacutelicos

asociados a la degradacioacuten Por ejemplo el aislamiento e identificacioacuten en el capiacutetulo 2 de

especies del geacutenero Nitrobacteria podriacutea estar relacionada con la reduccioacuten de nitritos

presentes en el medio contaminado (capiacutetulo 2 Gonzaacutelez et al 2010)

Los resultados de identificacioacuten mediante teacutecnicas moleculares muestran la evidente

variedad de geacuteneros implicados en estos procesos La variabilidad observada fue mucho

menos intensa con las teacutecnicas moleculares dependientes de cultivo (capiacutetulo 2) ya que tan

solo entre un 1 y un 10 del total de bacterias del suelo son cultivables (Nannipieri et al

2003) Algunos autores (Menn et al 1993 Okpokwasili et al 1986) proponen que los genes

cataboacutelicos para la degradacioacuten de HAP probablemente se transmitan horizontalmente

mediante plaacutesmidos entre bacterias pertenecientes a grupos taxonoacutemicos muy diferentes

Mueller et al (1997) afirman que no es probable encontrar una clara relacioacuten entre grupos

taxonoacutemicos de bacterias y la produccioacuten de enzimas implicadas en la degradacioacuten de

hidrocarburos aromaacuteticos Por tanto no es extrantildeo que genes homoacutelogos (en este caso

degradativos) se expresen en bacterias que taxonoacutemicamente estaacuten muy poco relacionadas

(capiacutetulos 2 3 y 4) otorgando una alta variabilidad al conjunto de bacterias con capacidad

degradadora


178

Preexposicioacuten a los HAP iquestes necesaria para una biodegradacioacuten eficaz HAP

Tradicionalmente algunos autores han sugerido que la preexposicioacuten de bacterias a un

determinado contaminante es necesaria para la adaptacioacuten y consecuente degradacioacuten

(Spain amp van Veld 1983) o para un aumento en la tasa del proceso (Haritash amp Kaushik

2009) Sin embargo autores como Johnsen amp Karlson (2005) se plantean si eacutesta es una

capacidad presente en las comunidades microbianas independientemente de su previa

exposicioacuten o si es una capacidad inducida por la exposicioacuten a elevados niveles de

contaminante Los procesos de biodegradacioacuten realizados con consorcios bacterianos

procedentes de zonas libres de contaminacioacuten por HAP (capiacutetulos 3 y 4) indican que eacutesta

es una capacidad intriacutenseca en las bacterias e independiente de la previa exposicioacuten y que

se manifiesta ante un periodo de contaminacioacuten Los geacuteneros identificados en el capiacutetulo 3

(Ralstonia Pseudomonas o Bacillus) son propios de sistemas con un alto contenido en

madera en descomposicioacuten en los que contribuyen a la degradacioacuten de lignocelulosa

celulosa y sus subproductos mediante enzimas oxidativas (Rastogi et al 2009) Las

enzimas lignoliacuteticas de los hongos saproacutefitos degradan compuestos con estructuras

quiacutemicas similares a la lignina como son los HAP (Hatakka 1994 2001 Barr amp Aust 1994

Meulenberg et al 1997) Por tanto las especies bacterianas con la bateriacutea enzimaacutetica para

degradar subproductos de lignina y celulosa pueden tambieacuten adaptarse y metabolizar HAP

(Tian et al 2008 Couling et al 2010) La capacidad degradadora de este tipo de

compuestos no solo se transmite de manera vertical sino que la transferencia horizontal de

genes puede ser tambien un factor determinante para la adquisicioacuten de esta capacidad entre

los microorganismos del consorcio o comunidad

Los resultados referentes a la alta capacidad degradativa que muestra el consorcio

BOS08 procedente de una zona limpia (capiacutetulo 3) gozan de una extraordinaria importancia

a nivel aplicado y ecoloacutegico La mayoriacutea de los trabajos que estudian el posible paralelismo

entre la degradacioacuten de lignina y la de compuestos aromaacuteticos se han llevado a cabo con

hongos maderables de la llamada ldquopodredumbre blancardquo El hecho de que un consorcio

bacteriano no adaptado a la degradacioacuten de HAP sea capaz de metabolizar eficazmente

HAP de alto peso molecular y ademaacutes disminuya la toxicidad del medio por debajo del

umbral de la toxicidad incluso a bajas temperaturas sin duda abre un campo de

investigacioacuten muy amplio dentro de la biorremediacioacuten Ecofisioloacutegicamente hablando

resultan tambieacuten de gran intereacutes estas espcies que han sido capaces de readaptar su

bateriacutea enzimaacutetica para metabolizar una fuente de carbono altamente recalcitrante y toacutexica

que no estaba presente en su medio natural


179

Posibles actuaciones en un medio contaminado

Ante un caso de contaminacioacuten de un medio con HAP o sustancias que los contengan la

biorremediacioacuten resulta una de las teacutecnicas maacutes eficaces y respetuosas con el medio La

atenuacioacuten natural del contaminante por los microorganismos presentes en el propio medio

depende seguacuten Frosyth et al (1995) del tamantildeo de la poblacioacuten degradadora autoacutectona No

obstante los resultados obtenidos durante el proceso de biorremediacioacuten de un suelo

contaminado con creosota (capiacutetulo 4) indican que los resultados que puede ofrecer la

atenuacioacuten natural no dependen uacutenicamente del nuacutemero inicial de microorganismos

degradadores Las pruebas realizadas indicaron en el momento que se produjo la

contaminacioacuten la actividad degradadora era nula aunque tras un corto peridodo de

exposicioacuten al mismo comenzoacute la actividad de los microorganismos degradadores Esto

quiere decir que aunque en un primer momento la poblacioacuten degradadora sea miacutenima la

presencia del contaminante favorece su dominancia y hace patente su capacidad

degradadora Ademaacutes hay que tener en cuenta varias de las cuestiones abordadas en

apartados previos como son la rapidez y facilidad que tienen los microorganismos para

transferir esta capacidad incluso entre diferentes taxas (Menn et al 1993) o la alta

adaptabilidad a una nueva fuente de carbono Sin embargo la atenuacioacuten natural es una

teacutecnica muy ventajosa porque evita dantildeos en la comunidad bacteriana del medio a

diferencia de cualquier otra actuacioacuten que en menor o mayor medida modifican las

condiciones originales del ecosistema

Uno de los principales problemas de la biorremediacioacuten es el tiempo necesario para

la completa eliminacioacuten del contaminante del medio para lo cual se han desarrollado

estrategias de actuacioacuten con el uacutenico objetivo de acelerar y mejorar la eficacia del proceso

La bioestimulacioacuten tiene como principal objetivo potenciar la capacidad degradadora de los

microorganismos degradadores autoacutectonos mediante la adicioacuten de nutrientes inorgaacutenicos al

medio contaminado Sin embargo los resultados referentes a esta teacutecnica no son

concluyentes dada la elevada variabilidad de los mismo Los casos en los que la

bioestimulacioacuten favorece el proceso de biodegradacioacuten estaacuten estrechamente relacionados

con el impedimento de que los nutrientes se conviertan en un factor limitante para los

microorganismos ya que tal y como expone Leys et al (2005) los requerimientos de

nitroacutegeno y foacutesforo aumentan notablemente durante un episodio de contaminacioacuten Sin

embargo son numerosos los estudios que han obtenido resultados desfavorables con esta

teacutecnica debido a cuestiones relacionadas con altos iacutendices de salinidad (Braddock et al

1997) o tal y como se observa en el capiacutetulo 4 con los cambios que la bioestimulacioacuten

genera en la comunidad bacteriana que en muchos casos inducen a relaciones negativas

entre las especies de la comunidad debido a la competencia por los nutrientes (Rolling-


180

Willfred et al 2002) Por otra parte la bioestimulacioacuten durante el proceso de

biorremediacioacuten del suelo contaminado con creosota (capiacutetulo 4) no favorecioacute

significativamente la biodegradacioacuten del contaminante Estos resultados se pueden atribuir a

una concentracioacuten de nutrientes suficientes en el medio o bien a la raacutepida y efectiva

capacidad degradativa de creosota que mostraron los microorganismos autoacutectonos

El bioaumento es una teacutecnica que pretende incrementar la eficacia del proceso de

biorremediacioacuten mediante el inoacuteculo a la poblacioacuten autoacutectona de una poblacioacuten

degradadora previamente definida como tal Sin embargo es un tratamiento complejo cuyos

resultados dependen de algo tan desconocido y variable como son las relaciones entre

especies y comunidades (Yu et al 2005) Por ello no hay gran nuacutemero de artiacuteculos en los

que se describan resultados favorables de esta teacutecnica pero podemos resumir que las

consecuencias del bioaumento dependen fundamentalmente de dos cuestiones Una de

ellas es que las relaciones de competencia que se establecen entre la comunidad

introducida y autoacutectona sean negativas para la poblacioacuten degradadora (Vintildeas et al 2005

Yu et al 2005) relaciones que principalmente estaacuten dirigidas por la competicioacuten por los

recursos (Yu et al 2005) La inexistencia de efectos significativos del bioaumento durante el

proceso de bodegradacioacuten de creosota (capiacutetulo 4) al igual que los descritos por Herwignen

et al (2005) indican que la presencia de una comunidad bacteriana autoacutectona con

capacidad degradadora o que se adapta raacutepidamente como es nuestro caso puede ser otra

de las cuestiones que hagan que el bioaumento no favorezca el proceso

Los ensayos de biorremediacioacuten realizados durante la presente tesis y los

consultados en las diferentes referencias bibliograficas nos llevan a concluir una vez maacutes

que los efectos de las diferentes teacutecnicas de biorremediacioacuten dependen de las condiciones

del medio contaminado y de la poblacioacuten bacteriana que alberga Por ello un estudio previo

que indique las caracteriacutesticas bioacuteticas (capacidad degradadora composicioacuten y evolucioacuten de

la comunidad bacteriana) y abioacuteticas (temperatura ambiente y caracteriacutesticas fisico-quiacutemicas

del suelo) del mismo asiacute como un breve ensayo a escala de laboratorio donde se apliquen

las diferentes teacutecnicas y seleccionando las maacutes adecuadas mejora en gran medida la

efectividad de la biorremediacioacuten in situ

Conclusiones generales

III


183


De los trabajos llevados a cabo en esta tesis doctoral se pueden extraer las siguientes

conclusiones generales

1 La optimizacioacuten de los factores abioacuteticos y bioacuteticos hace que el proceso de

biodegradacioacuten sea maacutes eficaz y permite modificarlo mediante estrategias de

biorremediacioacuten

2 Los factores que realmente influyen significativamente en un proceso se observan

mediante un estudio ortogonal de los mismos porque permite evaluar las

interacciones entre los factores seleccionados

3 No todos los ambientales son limitantes para la degradacioacuten de HAP Po ejemplo la

bioestimulacioacuten con nutrientes inorgaacutenicos no es efectiva en casos en los que la

cantidad de nutrientes en el medio es suficiente La adicioacuten de glucosa como fuente

adicional de carbono no es necesaria cuando el consorcio esta adaptado a los HAP

como fuente de carbono

4 Las actuaciones basadas en la adicioacuten de fuentes de carbono adicionales a los HAP

no son efectivas cuando los consorcios o cepas bacterianas estaacuten adaptadas a los

HAP porque esto supone un periodo de readaptacioacuten

5 La fuente de carbono disponible en cada momento durante un proceso de

biodegradacioacuten de HAP y otras sustancias en el medio como los surfactantes

condicionan la presencia de especies y por tanto la sucesioacuten de las mismas

6 La aparicioacuten de nuevas especies previamente no descritas como degradadoras

puede estar relacionada con la transferencia horizontal de genes degradativos que

en muchos casos ocurre entre grupos taxonoacutemicos poco relacionados lo que

ampliariacutea auacuten maacutes la expresioacuten de la capacidad degradativa en la comunidad

7 La identificacioacuten en un consorcio procedente de una zona limpia y rica en materia

orgaacutenica de especies fundamentales en sistemas de degradacioacuten de madera

sugiere que las enzimas oxidativas usadas por estas especies en la degradacioacuten de

subproductos de lignina y celulosa se emplean en la degradacioacuten de HAP Por tanto


184

la previa exposicioacuten de los consorcios bacterianos o cepas individuales a un

contaminante no es necesaria cuando tienen una bateriacutea enzimaacutetica que se puede

adaptar y metabolizar el contaminante

8 El papel de las especies bacterianas adaptadas a la degradacioacuten de HAP en

ambientes friacuteos (Tordf lt 15ordmC) es fundamental para la biorremediacioacuten en climas

extremos Ademaacutes la actividad de estas especies en periodos de bajas temperaturas

permite el crecimiento de otras especies de la comunidad bacteriana a partir de los

subproductos de degradacioacuten

9 El bioaumento es una teacutecnica cuyos resultados estaacuten ampliamente influenciados por

las relaciones que se establecen entre la comunidad autoacutectona e introducida y soacutelo

se recomienda en aquellos casos en los que la comunidad autoacutectona no tenga

microorganismos degradadores o no sean capaces de desarrollar esta capacidad

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IV


187


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Scott Base Antarctica Polar Biol 23 183-188




Atlas RM amp Bartha R 1972 Biodegradation of petroleum in seawater at low temperatures


Baek KH Yoon BD Kim BH Cho DH Lee IS Oh HM amp Kim HS 2007 Monitoring of

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Barkay T amp Pritchart H 1988 Adaptation of aquatic microbial communities to pollutant

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Barr DP amp Aust SD 1994 Mechanisms with rot fungi use to degrade pollutants Environ

Sci Technol 28 78A-87A










Braddock JF Ruth ML Catterall PH Walworth JL amp McCarthynd KA 1997

Enhancement and inhibition of microbial activity in hydrocarbon contaminated arctic

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2078-2084

Cavicchioli R Siddiqui KS Andrews D amp Sower KR 2002 Low temperature

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Cerniglia1984 Microbial metabolism of polycyclic aromatic hydrocarbons Adv Appl

Microbiol 30 31-71

Cerniglia 1992 Biodegradation of polycyclic aromatic hydrocarbons Biodegradation 2-3

351-368


188

Chaicircneau CH Morel J Dupont J Bury E amp Oudot J 1999 Comparison of the fuel oil

biodegradation potential of hydrocarbon assimilating microorganisms isolated from a

temperate agricultural soil Sci Total Environ 227 237-247

Chaicircneau CH Rogeus G Yeacutepreacutemian C amp Outdot J 2005 Effects of nutrients

concentration on the biodegradation of crude oil and associated microbial

populations in the soil Soil Biol Biochem 37 1490-1497

Chauhan A Fazlurrahman Oakeshott JG amp Jain RK 2008 Bacterial metabolisms of

polycyclic aromatic hydrocarbons strategies for remediation Indian J Microbiol

4895-113

Chen S-H amp Aitken MD 1999Salicylate stimulates the degradation of high-molecular


Environ Sci Technol 33 435ndash439

Chen J Wong MH amp Tam N 2008 Multi-factors on biodegradation kinetics of polycyclic



Cheung P-Y amp Kinkle BK 2005 Effect of nutrients and surfactant on pyrene mineralization

and Mycobacterium spp populations in contaminated soil Soil Biol Biochem 37

1401-1405



Clements WH Oris JT amp Wissin TE 1994 Accumulation and food chain transfer of

fluoranthene and benzo[a]pyrene in Chironomus riparius and Leponis macrochirus

Arch Environ Contam Toxicol 26261-266

Colwell RR Mills AL Walker JD Garcia Tello P amp Campos V 1978 Microbial ecological

studies at the Metula spill in the Strait of Magellan J Fish Res Board Can 35573-

580


chemical structure concentration and multiple amendment Environ Poll 158 3411-

3420

Das K amp Mukherjee AK 2006 Crude petroleum-oil biodegradation efficiency of Bacillus

subtillis and Pseudomonas aeruginosa strains isolated from a petroleum-oil

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Delille D amp Pelletier E 2002 Natural attenuation of diesel-oil contamination in a subantartic

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189


Phytoremediation of small-scale oil spills in fresh marsh environments a mesocosms



on hydrocarbon biodegradation in artic tundra soil Appl Environ Microbiol 67 5107-

5112




Microbiol 69 275-84

Felsenstein J 1985 Confidence limits on phylogenies an approach using the bootstrap

Evolution 39 783-791

Fiechter A 1992 Biosurfactants moving towards industrial application Trends Biotechnol

10 208-217

Fritsche JD 1985 Nature and significance of microbial cometabolism of xenobiotics J

Basic Bacteriol 25 603-619

Forsyth JV Tsao YM amp Bleam RD 1995 Biorremediation when is augmentation needed

In Hinchee RE Fredrickson J amp Alleman BC (Eds) Bioaugmentation for site

remediation Battelle Press Columbus pp 1-14

Ghazali FM Rahman RNZA Salleh AB amp Basr M 2004 Degradation of hydrocarbons





Grimberg SJ Stringfellow WT amp Aitken MD 1996 Quantifying the biodegradation of

phenanthrene by Pseudomonas stutzeri P16 in the presence of a nonionic surfactant


Habe H amp Omori T 2003 Gentics of polycyclic aromatic hydrocarbon metabolisms in

diverse aerobic bacteria Biosci Biotechnol Biochem 67 225-243

Haritash AK amp Kaushik CP 2009 Biodegradation aspects of polycyclic aromatic

hydrocarbons (PAHs) A review J Hazard Mater 169 1-15


role in lignin degradation FEMS Microbial Rev 13 125-135

Hatakka A 2001 Biodegradation of lignin In Hofrichter M amp Steinbuchel A (eds)


Germany p129-180

Herwijnen R Joffe B Ryngaert A Hausner M Springael D Govers HAJ Wuertz S amp



190



Internacional Agency for Research on Cancer 1972-1990 Monographs on the evaluation of

carcinogenics risks on human International Agency for Research on Cancer Lyons

France



Johnsen AR Wick LY amp Harms H 2005 Principles of microbial PAH-degradation in soil

Environ Pollut 133 71-84

Johnsen AR Lipthay JR Sorensen SJ Ekelund F Christensen P Andersen O

Karlson U amp Jcobsen CS 2006 Microbial degradation of street dust polycyclic

aromatic hydrocarbons in microcosms simulating diffuse pollution of urban soil

Environ Microbiol 8535-545





Kanaly RA amp Harayama S 2000 Biodegradation of high molecular weight polycyclic

aromatic hydrocarbons by bacteria J Bacteriol 182 2059-2067

Kaumlstner M Breueer-Jammali M amp Mahro B 1998 Impact of inoculation protocols salinity

and pH on the degradation of polycyclic aromatic hydrocarbons (PAHs) and survival

of PAH-degrading bacteria introduced into soil Appl Environ Microbiol 64 359-362

Kim Y H Freeman J P Moody J D Engesse K H amp Cerniglia C E 2005 Effects of pH

on the degradation of phenanthrene and pyrene by Mycobacterium vanbaalenii

PYR-1 Appl Environ Microbiol 67 275ndash285

Koeber R Bayona JM amp Niessner R 1999 Determination of benzene[a]pyrene diones in

air particulates matter with liquid chromatography mass spectrometry Environ Sci

Technol 33 1552-1558





Lee ML Novotny MV amp Bartle KD 1981 Analytical chemistry of polycyclic aromatic

hydrocarbons Academic Press Inc New York NY


biodegrdation by Pseudomonas putida G7 J Hazard Mater 105 157-167


191


carbonnitrogenphosphorus ratio on polycyclic aromatic hydrocarbons degradation by

Mycobacterium and Sphingomonas in soil Appl Microbiol Biotechnol 66 726-736

Lim LH Harrison RM amp Harrad S 1999 The contribution of traffic to atmospheric

concentration of polycyclic aromatic hydrocarbons Environ Sci Technol 33 3538-

3542

Liu Y Zhu L amp Shen X 2001 Polycyclic aromatic hydrocarbons in indoor and outdoor air of

Hangzhou China Environ Sci Technol 35 840-844



Maliszewska-Kordybach B 1996 Polycyclic aromatic hydrocarbons in agricultural soils in

Poland preliminary proposals for criteria to evaluate the level of soil contamination

Appl Geochem 11 212-127



3127-3133




degradation and enzyme activities of cold-adapted bacteria and yeasts

Extremophiles 7451ndash458

Martiacuten Guirao L 2007 Aproximacioacuten ecotoxicoloacutegica a la contaminacioacuten por metales

pesados en la laguna costera del Mar Menor Tesis doctoral Universidad de Murcia

Murcia

Menn F-M Applegate BM amp Sayler GS 1993 NAH-plasmid mediated catabolisms of

anthracene and phenanthrene to naphtoic acids Appl Environ Microbiol 59 1938-

1942

Meulenberg R Rijnaarts HHM Doddema HJ amp Field A 1997 Partially oxidized polycyclic

aromatic hydrocarbons show an increased bioavailability and biodegradability FEMS

Microbiol 152 45-49








192




Mueller JG Chapman PJ Blattman BO amp Pritchard PH 1990 Isolation and

characterization of a fluoranthene-utilizing strain of Pseudomonas paucimobilis Appl


Mueller JG Devereux R Santavy DL Lantz SE Willis SG amp Pritchard PH 1997

Phylogenetic and Physiological comparisions of PAH-degrading bacteria from

geographically diverse soils A van Leeuw J Microb 71 329-343


Microbial diversity and soil functions European J Soil Sci 54 655-670

Okpokwasili GC Somerville CC Grimes DJ amp Colwell RR 1986 Plasmid-associated

phenanthrene degradation by Chesapeake Bay sediment bacteria A Colloq Inst

Fran Rech Exploit Mer 3 601ndash610

Pelz O Tesar M Wittich RM Moore ERB Timmis KN Abraham WR 1999 Towards

elucidation of microbial community metabolic pathways unrevealing the network of

carbon sharing in a pollutant-degrading bacterial consortium by immunocapture and

isotopic ratio mass spectrometry Environ Microbiol 1167ndash174

Portaels F amp Pattyn SR 1982 Growth of mycobacteria in relation to the pH of the medium

Ann Microbiol 133 213-221

Puntus IF Filonov AE Akhmetov LI Karpov AV amp Boronin AM 2008 Phenanthrene

degradation by bacteria of the genera Pseudomonas and Burkholderia in model soil

systems Microbiology 77 7-15

Rastogi G Muppidi GL Gurram RN Adhikari A Bischoff KM Hughes SR Apel WA

Bang SS Dixon DJ amp Sani RK 2009 Isolation and characterization of cellulose-

degrading bacteria from the deep subsurface of the Homestake gold mine Lead

South Dakota USA J Ind Microbiol Biotechnol 36 585-598

Readman J W Fillmann G Tolosa I Bartocci J Villeneuve J -P Catinni C amp Mee L D

2002 Petroleum and PAH contamination of the Black Sea Marine Pollut Bull 44

48-62

Rolling Willfred FM Milner MG Jones DM Lee K Danniel F Swanell Richard JP amp

Head IM 2002 Robust hydrocarbons degradation and dynamics of bacterial

communities during nutrients-enhanced oil spill bioremediation Appl Environ

Microbiol 68 5537-5548

Rosenberg E amp Ron EZ 1999 High ndash and low- molecular mass microbial surfactant Appl

Microiol Biotechnol 52 154-162


193

Santos E C Jacques R J S Bento F M Peralba M-C R Selbach PA Saacute E L S

Camargo FAO 2008 Anthracene biodegradation and surface activity by an iron-

stimulated Pseudomonas sp Bioresource Technol 99 2644-2649


Shuttleworth KL amp Cerniglia E 1995 Environmental aspect of PAH biodegradation Appl

Biochem Biotechnol 54 291-302

Soberon-Chavez G Lepine F amp Deziel E 2005 Production of rhamnolipids by

Pseudomonas aeruginosa Appl Microbiol Biotechnol 68 718-725

Soriano JA Vintildeas MA Franco JJ Gonzaacutelez JM amp Albaigeacutes J 2006 Spatial and

temporal trends of petroleum hydrocarbons in wild mussels from the Galician coast

(NW Spain) affected by the Prestige oil spill Sci Total Environ 370 80-90








communities Aquat Microb Ecol 47 1-10

Tian L Ma P amp Zhong J-J 2003 Impact of presence of salicylate or glucose on enzyme

activity and phenanthrene degradation by Pseudomonas mendocina Process

Biochem 38 1125-1132



Xiamen China Marine Pollut Bull56 1184-1191


bacteria isolated from waste crude oil with polycyclic aromatic hydrocarbons

removal capacities Syst Appl Microbiol 29 244-252

Torres LG Rojas N Bautista G amp Iturbe R 2005 Effect of temperature and surfactantacutes

HLB and dose over the TPH-diesel biodegradation process in aged soils Process

Biochem 40 3296-3302





creosote-contaminated Soil App Environ Microbiol 71 7008-7018

Wagrowski DM amp Hites RA 1997 Polycyclic aromatic hydrocarbons accumulation in urban

suburban and rural vegetation Environ Sci Technol 31 279-282


194


cultures of Bacilus subtilis during biosurfactant fermentation J Biosci Bioeng 96

174-178



Pollut 139 1-13

Wu SC amp Gschwend PM 1986 Sorption kinetics of hydrophobic organic compounds to

natural sediments and soil Environ Sci Technol 20 717-725

Ye B Siddigi MA Maccubbin AE Kumar S amp Sikka HC 1996 Degradation of

polynuclear aromatic hydrocarbons by Sphyngomonas paucimobilis Environ Sci

Technol 30136-142

Yu KSH Wong AHY Yau KWY Wong YS amp Tam NFY 2005 Natural attenuation



Zender M 1983 Physical and chemical properties of polycyclic aromatic hydrocarbons p 1-

26 In ABjorseth (ed) Handbook of polycyclic aromatic hydrocarbons Marcel

Dekker Inc New York NY

Zhang XX Cheng SP Zhu CJ amp Sun SL 2006 Microbial PAH-degradation in soil

degradation pathways and contributing factors Pedosphere 16 555-565

Zhang Z Gai L Hou Z Yang C Ma C Wang Z Sun B He X Tang H amp Xu P 2010

Characterization and biotechnological potential of petroleum-degrading bacteria

isolated from oil-contaminated soils Bioresource Technol 101 8452ndash8456

Agradecimientos

197

Agradecimientos

Todaviacutea recuerdo el primer diacutea que entre en el departamento en el laboratorio

aquello me parecioacute todo un mundo en el cual sin quererlo y sin estar convencida de

ello acabeacute metida de lleno Poco a poco fueron pasando los meses los antildeos

presenteacute el Practicum y me diacute cuenta de que queriacutea seguir adelante Unos cuantos

antildeos despueacutes he llegado a la meta lo cual no hubiera sido posible sin mucha gente

que me ha apoyado ayudado y empujado en los momentos en los que yo no podiacutea

maacutes A todos ellos gracias por hacer que esto haya sido posible

El primero de mis agradecimientos va dirigido a Natalia Fernando y Mari

Carmen Os tengo que dar las gracias por brindarme la oportunidad de formar parte

del grupo y por tantas cosas que con cada uno de vosotros he aprendido Despueacutes

de estos antildeos creo que hemos conseguido cosas maravillosas aunque hayamos

tenido imprevistos de todo tipo porque como ya sabemos si ponemos un circo nos

crecen los enanoshellippero aquiacute estamos Somos un grupo muy equilibrado

profesionalmente tenemos un poco de todo y por otro lado no es mal balance tres

histeacutericas frente a uno que pone la calma no se como no hemos acabado contigo

Fernando Natalia gracias por hacerme controlar el miura que llevo dentro y tener

tanta paciencia Carmen gracias por estar siempre ahiacute ya sea desde Espantildea desde

el otro lado del charco o nada mas ser mamaacute En todo momento a nivel personal y

profesional me he sentido arropada por vosotros gracias por vuestro apoyo y ganas

de seguir adelante Vosotros habeis sido los responsables de que quiera investigar

Si una persona en concreto se merece especial agradecimiento es mi Yoli

Aunque al principio de todo no nos conociacuteamos no hay mas que vernos ahora Por

un lado ha sido imprescindible la ayuda que me has dado trabajando cuando maacutes

perdida estaba Por todo tu apoyo ten presnete que me has ayudado a escribir cada

una de las liacuteneas que has leiacutedo Has sabido ser mi amiga y estar conmigo cuando

maacutes lo he necesitado y hacer que me olvidara de todo ya sea haciendo toriijas

pizzas viendo una peli tomando una copichuela o con nuestros preciados pinchitos

sobre todo estos uacuteltimos meses estresantes en los que no seacute como no te he vuelto

loca Gracias tambieacuten por hacerme reir hasta llorar por preocuparte cada diacutea de

198

estas uacuteltimas semanas de coacutemo voy por conocerte todas y cada una de las cosas

en las que estoy trabajando y un largo etc Te conoces mejor yo el estado de cada

uno de mis artiacuteculos las correcciones que tengo y las que me faltan Eres estupenda

y espero no dejar de descubrir nunca cosas sobre ti Mil gracias

Son muchas las personas que han pasado por el despacho Pepe aunque

estas muy muy lejos agardezco tu ayuda continua sobre todo en el Maacutester la mitad

de las cosas se me hubieran olvidado si no hubiera sido por ti que cabeza la miacutea

Tambieacuten tengo que recordar a Raquel Felipe y Cris el antiguo equipo Ecotox

pasamos muy buenos ratos que se echan de menos A mis actuales compantildeeros

Alfredo Pesca Julia Silvia y Carlos que aunque no estas en el despacho como si lo

estuvieras Gracias por amenizar las horas de laboratorio y los madrugones Silvia

especialmente a ti gracias por sacar siempre un rato para charlar y escucharnos

mutuamente aunque nos separen un porroacuten de cajas y un poto gigante ahiacute estas

siempre Espero seguir aquiacute mucho tiempo para apoyarte igual que tuacute lo has hecho

conmigo Cris no me olvido de ti que desde el principio y hasta ahora te has

preocupado de saber que tal me iba estabas al tanto de todo y me has animado a

seguir adelante Te deseo que las cosas te vayan genial porque te lo mereces

asique aacutenimo que no es por presionar pero en breve te toca a tiacute Me faltan palabras

para contar todo lo que Moacutenica y Andrea me han ayudado y ensentildeado desde un

primer momento Igualmente agredezco el apoyo que Patri y Ester me han dado al

igual que los buenos ratos cotilleando imprescindibles Tambieacuten tengo que

agradecer a Jose Luis Sanz de la Universidad Autoacutenoma de Madrid que me abriera

las puertas de su laboratorio para aprender la maravillosa teacutecnica del DGGE y unas

cuantas cosas maacutes Ine a tiacute si que te agradezco un montoacuten las horas que has

perdido de tu trabajo para ensentildearme Desde un primer momento simpre con la

sonrisa puesta auacuten sin concocerme de nada Han sido muchos los viajes que he

hecho y el tiempo empleado en ello pero ha merecido la pena Asique igualmente

formas parte de esta tesis porque cada uno de los artiacuteculos no hubieran estado

completos sin tu ayuda

Son muchas las personas que sin formar parte del gremio han estado siempre

presentes Mis padres y mi hermano ya sabeis que no podriacutea hacer nada sin

vosotros a mi lado Durante estos antildeos no habeis dejado de preocuparos por mi y de

apoyarme ni un solo instante maacutes auacuten cuando las cosas han sido tan complicadas

199

para miacute Nada hubiera salido bien sin vosotros una vez maacutes os doy las gracias por

ser tan maravillosos y por teneros Por otro lado mis amigos ellos si que andan

agenos al tema y sin embargo siempre han sabido cuando preguntar y que palabras

usar cuando mas lo he necesitado Tengo que hacer una mencioacuten especial al sentildeor

Jimmy (responsable graacutefico y de disentildeo de la tesis) a ti si que te ha caiacutedo una

buena desde aquella llamada en la que te dije ldquoha llegado el momentordquo A

parte del gran trabajo que has hecho vistiendo la tesis tuacute una de las personas maacutes

sosegadas que conozco has podido aguantar el histerismo de los diacuteas previos a

depositar la tesis Gracias Jimmy eres un sol pero que sepas que auacuten me queda la

defensa oacutesea presentacioacuten en power pointhellipyo no digo nada Tambieacuten

agaradezco al ldquogrupo parkeeerdquo las horas que pasamos en las cuales a parte de

mucho friacuteo tambieacuten pasamos muy buenos ratos Especialmente a Lauri gracias por

acercarte un buen diacutea y preguntarme iquesttodo bien Desde entonces tus achuchones

tus canciones y tu ldquoflower powerrdquo han hecho que me despeje cada tarde Gracias

tambieacuten por interesarte tanto por mi trabajo dentro de poco te lo podreacute presentar

Las uacuteltimas palabras van dirigidas a Javi A ti que has estado conmigo desde el

principio gracias por no dejar que me desquicie y darme siempre tranquilidad Son

muchas las horas que he dedicado a esto y siempre has estado recordaacutendome

cuando era el momeno de parar Gracias por saber comprender lo que hago aunque

a veces me queje tanto y por ayudarme a echarle un par de narices cuando maacutes

desanimada estaba Gracias por hacer que este mundo roto no estropee mi sonrisa

Todo el tiempo que no te he dedicado lo recuperaremos juntos en nuestra casa

A todos y cada uno de vosotros gracias

Raquel

Dra Natalia Gonzaacutelez y Dra Mariacutea del Carmen Molina profesoras titulares del

Departamento de Biologiacutea y Geologiacutea de la Universidad Rey Juan Carlos

CERTIFICAN

Que los trabajos de investigacioacuten desarrollados en la memoria de tesis doctoral

ldquoBiorremediacioacuten de suelos contaminados con hidrocarburos aromaacuteticos policiacuteclicosrdquo son aptos para ser presentados por la Lda Raquel Simarro Doblado ante el

Tribunal que en su diacutea se consigne para aspirar al Grado de Doctor en Ciencias

Ambientales por la Universidad Rey Juan Carlos de Madrid

VordmBordm Director Tesis VordmBordm Director de Tesis

Dra Natalia Gonzaacutelez Beniacutetez Dra Mordf Carmen Molina



Iacutendice















Resumen



I


13

Antecedentes






















contaminados








14

























15


1996)


























1500000









16











ambiental posible

















Temperatura y pH




17









Kaushik 2009)






























18




































19





































20





































21





































22




































23








Resumen Objetivos

25

Objetivos






























27
















JA









28


M






29





































30






bacteriano C2PL05
































31










contaminante

























33




















muerta




A B


34

















naturales











35


Optimizacioacuten

CNP

BHB tween-80


antraceno

X 3

100101

1002116

100505

Optimizacioacuten

fuente de N

BHB tween-80


antraceno

X 3

NaNO3

NH4NO3

(NH4)2SO3

Optimizacioacuten

fuente de Fe

BHB tween-80


antraceno

X 3

FeCl3

Fe(NO3)3

Fe2(SO4)3

Optimizacioacuten

[Fe]

BHB tween-80


antraceno

X 3

005 mM

01 mM

02 mM

Optimizacioacuten

pH

BHB tween-80


antraceno

X 3

50

70

80

Optimizacioacuten

fuente de C

BHB tween-80

C2PL05



X 3

HAP

HAPglucosa (5050)

Glucosa

2ordm 3ordm

4ordm 5ordm 6ordm



36

Tordf









1001011002116100505

NaNO3

NH4NO3

(NH4)2SO3

FeCl3Fe(NO3)3

Fe2(SO4)3

005 mM01 mM02 mM

CMC20 CMC

10-1

10-2

10-3

0100505020100

18 tratamientos

X 3












X 3

X 3



el capiacutetulo 2


37














38

Tratamiento 1

Tratamiento 2

Tratamiento 3

Tratamiento 4









X 3

X 3

X 3

X 3

X 5 tiempos

X 5 tiempos

X 5 tiempos

X 5 tiempos
















39







natural




y Bioaumento






Suelo natural +X 2

Suelo natural +X 2

Suelo natural +X 2

Suelo natural +X 2

Suelo natural +X 2




40





contaminado




pH - 77 plusmn 01


WHCa v 33 plusmn 7


















consorcio










41




























42













(ordmC)











43






Tordfhibridc


Tordf finalf

4 ordmC infin

95 ordmC 5 min

95 ordmC 1 min

54 ordmC 05 min

72 ordmC 15 min

72 ordmC 10 min

30 CICLOS

PROGRAMA PCR III


Tordfhibridc


Tordf finalf

4 ordmC infin

95 ordmC 9 min

94 ordmC 1 min

55 ordmC 1 min

72 ordmC 15 min

72 ordmC 5 min

30 CICLOS

PROGRAMA PCR II


Tordfhibridc


Tordf finalf

4 ordmC infin

94 ordmC 9 min

94 ordmC 1 min

55 ordmC 1 min

72 ordmC 15 min

72 ordmC 5 min

30 CICLOS

PROGRAMA PCR I


44


Escherichia coli








comunidad
























45



































Capiacutetulo







1a


49

Abstract





















and so the costs


51

Introduction



































52



degradation rate


Chemicals and media




























53














Experimental design


















growth


54




















Bacterial growth





equation

1

1

ii

iii tt





55

Kinetic degradation







iBiiAii

i CkCkdt

dCr Eq 2




each PAH








was inoculated









56

Results







0 100 200 300 400 500 600 700

20

40

60

80

100

Rem

aini

ng P

AH

(

)

Time (hour)





CDI kB









pH 2 30middot10-2 1103 4 15middot10-4 5






57













171819202122232425

100101 1002116100505

bb

a

A

CNP molar ratio

CD

I


-30

-25

-20

-15

-10

-05

00B

d

g

e

bc

f

ab

f

Log

k B (

h-1)





deviation


58








19

20

21

22

23

24

25

(NH4)

2SO

4NH4NO

3NaNO

3

a

b

a

A

Nitrogen source

CD

I


2x10-3

4x10-3

6x10-3

8x10-3

1x10-2

Bf

ba

e

bcb

dbc

a

kB (

h-1)






59











168

172

176

180

184

188

192

196

Fe(NO3)

3 Fe2(SO

4)

3FeCl

3

ab

b

a

A

Iron source

CD

I


2x10-3

4x10-3

6x10-3

8x10-3

1x10-2

B

c

a

b

c

b

d

b

a a

k B

(h-1

)






60










005 01 02

38

40

42

44

46

48

50

a

a

a

A


CD

I


50x10-3

10x10-2

15x10-2

20x10-2

B

c

f

d

b

e

d

cb

a

k B (

h-1)





standard deviation


61









5 7 8

215

220

225

230

235

240

245

a

b

a

A

pH

CD

I


50x10-3

10x10-2

15x10-2

20x10-2

25x10-2

30x10-2

B

b

a

ab ab

a

ab

c

ab ab

kB

(h-1

)






62










source

PAHs (100)


18

20

22

24

26

28

Carbon source

b

c

a

A

CD

I


2x10-2

4x10-2

6x10-2

8x10-2

1x10-1

B

c

bb

b

b

a

k B (h

-1)







63

Discussion


































64



























associated costs

Acknowledgements






65

References




Biodegr 63 913-922








13











269-281





1612-1614







66







Elsevier








Dordrecht pp 1-23




5548










174-178



Soil Poll 13 1-13



Capiacutetulo





experimental design



1b


69

Abstract









71

Introduction



































72


degradation
















Chemicals and media










73


Treatment T

(ordmC) CNP (molar)

N source

Fe

source


(mM)

Glucose PAH ()


Inoculum dilution

1 30 100505 (NH4)2SO3 Fe2(SO4)3 02 0100 CMC 10-3

2 20 1002116 (NH4)2SO3 FeNO3 005 0100 + 20CMC 10-2

3 25 100101 NaNO3 FeNO3 02 0100 + 20CMC 10-1

4 20 100505 NaNO3 Fe2(SO4)3 02 5050 + 20CMC 10-2

5 25 100505 NH4NO3 FeNO3 01 5050 CMC 10-2

6 30 100101 NH4NO3 Fe2(SO4)3 005 8020 + 20CMC 10-2

7 30 100101 NaNO3 FeCl3 01 0100 CMC 10-2

8 20 100505 NaNO3 FeCl3 005 8020 CMC 10-1

9 25 1002116 (NH4)2SO3 FeCl3 02 8020 CMC 10-2

10 20 1002116 NH4NO3 Fe2(SO4)3 01 0100 CMC 10-1

11 20 100101 NH4NO3 FeNO3 02 8020 CMC 10-3

12 25 100101 (NH4)2SO3 Fe2(SO4)3 005 5050 CMC 10-1

13 25 1002116 NaNO3 Fe2(SO4)3 01 8020 + 20CMC 10-3

14 30 1002116 NH4NO3 FeCl3 02 5050 + 20CMC 10-1

15 25 100505 NH4NO3 FeCl3 005 0100 + 20CMC 10-3

16 30 1002116 NaNO3 FeNO3 005 5050 CMC 10-3

17 30 100505 (NH4)2SO3 FeNO3 01 8020 + 20CMC 10-1

18 20 100101 (NH4)2SO3 FeCl3 01 5050 + 20CMC 10-3











Experimental design





74












Cell density





1

1

ii

iii tt















75






















76




each treatment


1 965 883 864 904 2 969 950 833 917 3 966 895 845 902 4 972 915 921 872 5 969 904 950 882 6 982 935 995 852 7 964 883 859 902 8 977 953 964 823 9 976 936 984 825 10 970 910 895 925 11 979 968 986 888 12 966 889 920 850 13 978 930 993 835 14 966 897 943 871 15 963 881 898 914 16 963 886 951 867 17 977 954 986 861 18 976 930 967 915



100

50

5

100

101

100

211

6

CNP

20

ordmC

25ordmC

30ordmC

82

84

86

88

90

92 T (ordmC)

aa

a

aa

aa

aa

a

Tot

al P

D (

)

NaN

O3

NH

4NO

3

(NH

4)2S

O3

N source

FeC

L3

FeN

O3

Fe2

(SO

4)3

a

a

0acute05 0acute1

0acute2

Fe source

a

a

a

0 -

100

50 -

50

80 -

20

C Fe (mM)

a

b

c

CM

C

+ 2

0 C

MC

Gluc-PAHs

aa

10^-

1

10^-

2

10^-

3DilutionCMC

aa

a





77





T (ordmC) Error

2 056 1889 2 22 183 ns

51 002 51 12


2 003 069 ns 2 22 183 ns

51 005 51 12

N source Error

2 001 007 ns 2 214 177 ns 51 005 51 121

Fe source Error

2 003 066 ns 2 89 071 ns

51 005 51 126


2 007 146 ns 2 118 095 ns 51 005 51 124

Glucose-PAH Error

2 024 584 2 1802

3085 51 004 51 395

8

CMC Error

1 001 027 ns 1 89 071 ns

52 005 52 125


2 331 a 2 113 091 ns 54 6614 51 125


median = 044

p-value lt 001

p-value lt 0001

100

50

5

100

100

1

100

211

6

CNP

20

ordmC

25ordmC

30ordmC

16

17

18

19

20

21

a

a

aa

a

aa

a

c

bCD

I

NaN

O3

NH

4NO

3

(NH

4)2S

O3

N source

FeC

L3

FeN

O3

Fe2

SO

4

Fe source

a

a

0acute05 0acute1

0acute2

C Fe (mM)

a

a

a

0-10

0

50-5

0

80-2

0

Gluc-PAH

a

b

c

CM

C

+ 2

0 C

MC

CMC

aa

10^-

1

10^-

2

10^-

3

00

05

10

15

20

25

30

35C

DI n

orm

aliz

ed

DilutionT (ordmC)

b

a

a





78
























solubility














79



































can grow vigorously

80









only PAH

Acknowledgements






81

References















5112






1612-1614







1380







8 315-323

82












Dordrecht pp 1-23

























Poll 139 1-13


83



4 252-258



Capiacutetulo







2


87

Abstract















89

Introduction




































90



























Chemicals and media








91

































92







is avoided

SK

S

S

max

(Equation 1)




Xdt

dX (Equation 2)

















93










PAH monitoring








iBiiAii

i CkCkdt

dCr (Equation 3)














94


































95




process



















code DUB











96






























97

00

02

04

06

08

10

12

14

16

18

0 5 10 15 20 25 30 35 40 45

30

40

50

60

70

80

90

100

Tox

icity

(

)

Time (day)

B

A

Abs

orba

nce 60

0 nm

(A

U)







98








0 5 10 15 20 25 30 35 40 45

0

10

20

30

40

50

60

70

80

90

100

Res

idua

l con

cent

ratio

n of

PA

Hs

()

Time (days)

















99







Error 0021 2


PAH 15middot10-4 2 779 0001





























100
























101










102















DUB Band














103


















104

0 15 30

0102030405060708090

100

102030405060708090

100

D

C

B

A

0 15 30

F DIC-1JA DIC-2JA

E

G DIC-6JA DIC-5JA

0 15 30

H

Time (day)


Pse

udom

onas

ribot

ypes

(

)


102030405060708090

100

Ste

notr

opho

mon

as

ribot

ypes

(

)

DIC-6JA

0 15 30

102030405060708090

100

Ent

erob

acte

r rib

otyp

es (

)

DIC-4RS

Time (days)

Tot

al s

trai

ns (

)








105


degradation




































106




















107












108




30 2469 19

24 881 3447

27 845

21 516










Conclusions











109

Acknowledgment








110

References



































111



212



213ndash221




839-844






























112







647-6554














Capiacutetulo







3


115

Abstract
















117

Introcuduction



































118
















of consortium used


Chemicals and media














119
































120

1

1

ii

iii tt
































121


































122


used as out-group


degrading process





























123









Results






obtained

0 5 10 15 20 25 30 35

BO S08

C 2PL05

tim e (m in)




124






















125

0 11 33 66 101 137

005

010

015

020

025

030

035

0 11 33 66 101 137

0 33 101 137102

103

104

105

106

107

108

109


Time (day)

Abs

orba

nce 6

00nm

(A

U)

Time (day)

DC

BA

cell

g so

il




temperature range


126





μ


C2PL05 H 158 b 09 C2PL05 L 105 a 17

BOS08 H 241 c 17

BOS08 L 189 b 12









Factor df SS F

p-value

A) μ



Error 8 49 x 10-5 0001



Error 8 1281



PAH d 1 85098 251


Error 16 54225





127





0 11 33 66 101 137

0

20

40

60

80

100

0 11 33 66 101 137

BA

Time (day)

Tox

icity

(

)

Time (day)

















128












129



















130




(genera) 33

C2PL05

15 ordmC-5 ordmC



25 ordmC-15 ordmC



BOS08

15 ordmC-5 ordmC



25 ordmC-15 ordmC



101

C2PL05

15ordmC-5ordmC



25 ordmC-15 ordmC



BOS08

15 ordmC-5 ordmC




131

25 ordmC-15 ordmC




biodegradation




























132







BOS08 consortium



by SIMPER method



22 DUB-25RS 2855 2789 2581 20 DUB-24RS 2993 2521 1797 2366


4 Unidentified 2855 1120 2362 1755 2315 175

27 Unidentified 139

2 Unidentified 1198

24 DUB-26RS 929




133






101


134





Discussion


















135











toxicity were high



























136




































137










Acknowledgements




138

References





Biodegr 63 913-922


















95-113





3420



361-368




139



675107-5112




Microbiol 69 275-84









Germany p129-180






















140

















3127-3133





7451ndash458






28 567












141







90









New York NY













15





157 174-209



42 252-258


142









Proteobacteria

Capiacutetulo







4


145

Abstract

















147

Introduction




































148


























Experimental design







149










microorganisms)










C2PL05

T5 Biostimulation

+ Bioaugmentation
















150

































151





































152

















136 and 145 days

Equation 2




Results











153

October

November

DecemberJanuary

FebruaryMarch

April MayJune

468

10121416182022

0 day

40 day

145 day

176 day

6 dayT

empe

ratu

re (

ordmC)

Month










6 40 145 17600

10x10-4

20x10-4

30x10-4

40x10-4

50x10-4

a a

b

aCO

2 mol

esg

of

soil

Time (days)



(plt005 SNK)


154

Toxicity assays











0 6 20 40 56 77 84 91 98 1051121251321411760

10

20

30

40

50

60

70

80

90

100 BA

Tox

icity

(

)


c

c

c

b

c

bc

bcbc

aa

aa

Treatment







155













156

T1 T2 T3 T4 T50000

0005

0010

0015

0020

0025

0030

0035

0040

g cr

eoso

te

g so

il


102030405060708090

100

C

aab

abb

a

bb

B

A

Ave

rage

res

idua

l con

cenr

atio

n of

PA

H (

)

T2 T3 T4 T50

102030405060708090

100

Tot

al r

esid

ual c

once

ntra

tion

of

PA

H (

)








157





Factor df SS F P




Error 20 14-5




Error 12 49143


PAH 2 168113 1452


Error 12 69486

p-value lt 005

p-value lt 001

p-value lt 0001


degradation















158













145 days


3 DUB-12RS

DUB-17RS 2875

16 DUB-17RS 1826

17 DUB-12RS

DUB-16RS 1414




176 days




26 DUB-13RS 1296




159






bands cloning


160



and 176 days (B)

















161

















162








163

Discussion















Colwell 1990)













contaminant







164






































165







166

References


































3411-3420


167







































168








3127-3133


























15





169




Orsis 13 105-117









42 252-258





Int 32 149-154





II


173



biodegradacioacuten
































174







































175



































176























1b)














177


































degradadora


178




































179






































180































III


183






























184














IV


187




























2078-2084




Microbiol 30 31-71


351-368


188









4895-113









1401-1405








580



3420









189






5112




Microbiol 69 275-84




10 208-217






















Germany p129-180




190





France























Technol 33 1552-1558










191






3542










3127-3133








Murcia



1942



Microbiol 152 45-49








192






























48-62








193






















Biochem 38 1125-1132









Biochem 40 3296-3302









194



174-178



Pollut 139 1-13





Technol 30136-142












Agradecimientos

197

Agradecimientos






























198


































199
























Raquel



Iacutendice















Resumen



I


13

Antecedentes






















contaminados








14

























15


1996)


























1500000









16











ambiental posible

















Temperatura y pH




17









Kaushik 2009)






























18




































19





































20





































21





































22




































23








Resumen Objetivos

25

Objetivos






























27
















JA









28


M






29





































30






bacteriano C2PL05
































31










contaminante

























33




















muerta




A B


34

















naturales











35


Optimizacioacuten

CNP

BHB tween-80


antraceno

X 3

100101

1002116

100505

Optimizacioacuten

fuente de N

BHB tween-80


antraceno

X 3

NaNO3

NH4NO3

(NH4)2SO3

Optimizacioacuten

fuente de Fe

BHB tween-80


antraceno

X 3

FeCl3

Fe(NO3)3

Fe2(SO4)3

Optimizacioacuten

[Fe]

BHB tween-80


antraceno

X 3

005 mM

01 mM

02 mM

Optimizacioacuten

pH

BHB tween-80


antraceno

X 3

50

70

80

Optimizacioacuten

fuente de C

BHB tween-80

C2PL05



X 3

HAP

HAPglucosa (5050)

Glucosa

2ordm 3ordm

4ordm 5ordm 6ordm



36

Tordf









1001011002116100505

NaNO3

NH4NO3

(NH4)2SO3

FeCl3Fe(NO3)3

Fe2(SO4)3

005 mM01 mM02 mM

CMC20 CMC

10-1

10-2

10-3

0100505020100

18 tratamientos

X 3












X 3

X 3



el capiacutetulo 2


37














38

Tratamiento 1

Tratamiento 2

Tratamiento 3

Tratamiento 4









X 3

X 3

X 3

X 3

X 5 tiempos

X 5 tiempos

X 5 tiempos

X 5 tiempos
















39







natural




y Bioaumento






Suelo natural +X 2

Suelo natural +X 2

Suelo natural +X 2

Suelo natural +X 2

Suelo natural +X 2




40





contaminado




pH - 77 plusmn 01


WHCa v 33 plusmn 7


















consorcio










41




























42













(ordmC)











43






Tordfhibridc


Tordf finalf

4 ordmC infin

95 ordmC 5 min

95 ordmC 1 min

54 ordmC 05 min

72 ordmC 15 min

72 ordmC 10 min

30 CICLOS

PROGRAMA PCR III


Tordfhibridc


Tordf finalf

4 ordmC infin

95 ordmC 9 min

94 ordmC 1 min

55 ordmC 1 min

72 ordmC 15 min

72 ordmC 5 min

30 CICLOS

PROGRAMA PCR II


Tordfhibridc


Tordf finalf

4 ordmC infin

94 ordmC 9 min

94 ordmC 1 min

55 ordmC 1 min

72 ordmC 15 min

72 ordmC 5 min

30 CICLOS

PROGRAMA PCR I


44


Escherichia coli








comunidad
























45



































Capiacutetulo







1a


49

Abstract





















and so the costs


51

Introduction



































52



degradation rate


Chemicals and media




























53














Experimental design


















growth


54




















Bacterial growth





equation

1

1

ii

iii tt





55

Kinetic degradation







iBiiAii

i CkCkdt

dCr Eq 2




each PAH








was inoculated









56

Results







0 100 200 300 400 500 600 700

20

40

60

80

100

Rem

aini

ng P

AH

(

)

Time (hour)





CDI kB









pH 2 30middot10-2 1103 4 15middot10-4 5






57













171819202122232425

100101 1002116100505

bb

a

A

CNP molar ratio

CD

I


-30

-25

-20

-15

-10

-05

00B

d

g

e

bc

f

ab

f

Log

k B (

h-1)





deviation


58








19

20

21

22

23

24

25

(NH4)

2SO

4NH4NO

3NaNO

3

a

b

a

A

Nitrogen source

CD

I


2x10-3

4x10-3

6x10-3

8x10-3

1x10-2

Bf

ba

e

bcb

dbc

a

kB (

h-1)






59











168

172

176

180

184

188

192

196

Fe(NO3)

3 Fe2(SO

4)

3FeCl

3

ab

b

a

A

Iron source

CD

I


2x10-3

4x10-3

6x10-3

8x10-3

1x10-2

B

c

a

b

c

b

d

b

a a

k B

(h-1

)






60










005 01 02

38

40

42

44

46

48

50

a

a

a

A


CD

I


50x10-3

10x10-2

15x10-2

20x10-2

B

c

f

d

b

e

d

cb

a

k B (

h-1)





standard deviation


61









5 7 8

215

220

225

230

235

240

245

a

b

a

A

pH

CD

I


50x10-3

10x10-2

15x10-2

20x10-2

25x10-2

30x10-2

B

b

a

ab ab

a

ab

c

ab ab

kB

(h-1

)






62










source

PAHs (100)


18

20

22

24

26

28

Carbon source

b

c

a

A

CD

I


2x10-2

4x10-2

6x10-2

8x10-2

1x10-1

B

c

bb

b

b

a

k B (h

-1)







63

Discussion


































64



























associated costs

Acknowledgements






65

References




Biodegr 63 913-922








13











269-281





1612-1614







66







Elsevier








Dordrecht pp 1-23




5548










174-178



Soil Poll 13 1-13



Capiacutetulo





experimental design



1b


69

Abstract









71

Introduction



































72


degradation
















Chemicals and media










73


Treatment T

(ordmC) CNP (molar)

N source

Fe

source


(mM)

Glucose PAH ()


Inoculum dilution

1 30 100505 (NH4)2SO3 Fe2(SO4)3 02 0100 CMC 10-3

2 20 1002116 (NH4)2SO3 FeNO3 005 0100 + 20CMC 10-2

3 25 100101 NaNO3 FeNO3 02 0100 + 20CMC 10-1

4 20 100505 NaNO3 Fe2(SO4)3 02 5050 + 20CMC 10-2

5 25 100505 NH4NO3 FeNO3 01 5050 CMC 10-2

6 30 100101 NH4NO3 Fe2(SO4)3 005 8020 + 20CMC 10-2

7 30 100101 NaNO3 FeCl3 01 0100 CMC 10-2

8 20 100505 NaNO3 FeCl3 005 8020 CMC 10-1

9 25 1002116 (NH4)2SO3 FeCl3 02 8020 CMC 10-2

10 20 1002116 NH4NO3 Fe2(SO4)3 01 0100 CMC 10-1

11 20 100101 NH4NO3 FeNO3 02 8020 CMC 10-3

12 25 100101 (NH4)2SO3 Fe2(SO4)3 005 5050 CMC 10-1

13 25 1002116 NaNO3 Fe2(SO4)3 01 8020 + 20CMC 10-3

14 30 1002116 NH4NO3 FeCl3 02 5050 + 20CMC 10-1

15 25 100505 NH4NO3 FeCl3 005 0100 + 20CMC 10-3

16 30 1002116 NaNO3 FeNO3 005 5050 CMC 10-3

17 30 100505 (NH4)2SO3 FeNO3 01 8020 + 20CMC 10-1

18 20 100101 (NH4)2SO3 FeCl3 01 5050 + 20CMC 10-3











Experimental design





74












Cell density





1

1

ii

iii tt















75






















76




each treatment


1 965 883 864 904 2 969 950 833 917 3 966 895 845 902 4 972 915 921 872 5 969 904 950 882 6 982 935 995 852 7 964 883 859 902 8 977 953 964 823 9 976 936 984 825 10 970 910 895 925 11 979 968 986 888 12 966 889 920 850 13 978 930 993 835 14 966 897 943 871 15 963 881 898 914 16 963 886 951 867 17 977 954 986 861 18 976 930 967 915



100

50

5

100

101

100

211

6

CNP

20

ordmC

25ordmC

30ordmC

82

84

86

88

90

92 T (ordmC)

aa

a

aa

aa

aa

a

Tot

al P

D (

)

NaN

O3

NH

4NO

3

(NH

4)2S

O3

N source

FeC

L3

FeN

O3

Fe2

(SO

4)3

a

a

0acute05 0acute1

0acute2

Fe source

a

a

a

0 -

100

50 -

50

80 -

20

C Fe (mM)

a

b

c

CM

C

+ 2

0 C

MC

Gluc-PAHs

aa

10^-

1

10^-

2

10^-

3DilutionCMC

aa

a





77





T (ordmC) Error

2 056 1889 2 22 183 ns

51 002 51 12


2 003 069 ns 2 22 183 ns

51 005 51 12

N source Error

2 001 007 ns 2 214 177 ns 51 005 51 121

Fe source Error

2 003 066 ns 2 89 071 ns

51 005 51 126


2 007 146 ns 2 118 095 ns 51 005 51 124

Glucose-PAH Error

2 024 584 2 1802

3085 51 004 51 395

8

CMC Error

1 001 027 ns 1 89 071 ns

52 005 52 125


2 331 a 2 113 091 ns 54 6614 51 125


median = 044

p-value lt 001

p-value lt 0001

100

50

5

100

100

1

100

211

6

CNP

20

ordmC

25ordmC

30ordmC

16

17

18

19

20

21

a

a

aa

a

aa

a

c

bCD

I

NaN

O3

NH

4NO

3

(NH

4)2S

O3

N source

FeC

L3

FeN

O3

Fe2

SO

4

Fe source

a

a

0acute05 0acute1

0acute2

C Fe (mM)

a

a

a

0-10

0

50-5

0

80-2

0

Gluc-PAH

a

b

c

CM

C

+ 2

0 C

MC

CMC

aa

10^-

1

10^-

2

10^-

3

00

05

10

15

20

25

30

35C

DI n

orm

aliz

ed

DilutionT (ordmC)

b

a

a





78
























solubility














79



































can grow vigorously

80









only PAH

Acknowledgements






81

References















5112






1612-1614







1380







8 315-323

82












Dordrecht pp 1-23

























Poll 139 1-13


83



4 252-258



Capiacutetulo







2


87

Abstract















89

Introduction




































90



























Chemicals and media








91

































92







is avoided

SK

S

S

max

(Equation 1)




Xdt

dX (Equation 2)

















93










PAH monitoring








iBiiAii

i CkCkdt

dCr (Equation 3)














94


































95




process



















code DUB











96






























97

00

02

04

06

08

10

12

14

16

18

0 5 10 15 20 25 30 35 40 45

30

40

50

60

70

80

90

100

Tox

icity

(

)

Time (day)

B

A

Abs

orba

nce 60

0 nm

(A

U)







98








0 5 10 15 20 25 30 35 40 45

0

10

20

30

40

50

60

70

80

90

100

Res

idua

l con

cent

ratio

n of

PA

Hs

()

Time (days)

















99







Error 0021 2


PAH 15middot10-4 2 779 0001





























100
























101










102















DUB Band














103


















104

0 15 30

0102030405060708090

100

102030405060708090

100

D

C

B

A

0 15 30

F DIC-1JA DIC-2JA

E

G DIC-6JA DIC-5JA

0 15 30

H

Time (day)


Pse

udom

onas

ribot

ypes

(

)


102030405060708090

100

Ste

notr

opho

mon

as

ribot

ypes

(

)

DIC-6JA

0 15 30

102030405060708090

100

Ent

erob

acte

r rib

otyp

es (

)

DIC-4RS

Time (days)

Tot

al s

trai

ns (

)








105


degradation




































106




















107












108




30 2469 19

24 881 3447

27 845

21 516










Conclusions











109

Acknowledgment








110

References



































111



212



213ndash221




839-844






























112







647-6554














Capiacutetulo







3


115

Abstract
















117

Introcuduction



































118
















of consortium used


Chemicals and media














119
































120

1

1

ii

iii tt
































121


































122


used as out-group


degrading process





























123









Results






obtained

0 5 10 15 20 25 30 35

BO S08

C 2PL05

tim e (m in)




124






















125

0 11 33 66 101 137

005

010

015

020

025

030

035

0 11 33 66 101 137

0 33 101 137102

103

104

105

106

107

108

109


Time (day)

Abs

orba

nce 6

00nm

(A

U)

Time (day)

DC

BA

cell

g so

il




temperature range


126





μ


C2PL05 H 158 b 09 C2PL05 L 105 a 17

BOS08 H 241 c 17

BOS08 L 189 b 12









Factor df SS F

p-value

A) μ



Error 8 49 x 10-5 0001



Error 8 1281



PAH d 1 85098 251


Error 16 54225





127





0 11 33 66 101 137

0

20

40

60

80

100

0 11 33 66 101 137

BA

Time (day)

Tox

icity

(

)

Time (day)

















128












129



















130




(genera) 33

C2PL05

15 ordmC-5 ordmC



25 ordmC-15 ordmC



BOS08

15 ordmC-5 ordmC



25 ordmC-15 ordmC



101

C2PL05

15ordmC-5ordmC



25 ordmC-15 ordmC



BOS08

15 ordmC-5 ordmC




131

25 ordmC-15 ordmC




biodegradation




























132







BOS08 consortium



by SIMPER method



22 DUB-25RS 2855 2789 2581 20 DUB-24RS 2993 2521 1797 2366


4 Unidentified 2855 1120 2362 1755 2315 175

27 Unidentified 139

2 Unidentified 1198

24 DUB-26RS 929




133






101


134





Discussion


















135











toxicity were high



























136




































137










Acknowledgements




138

References





Biodegr 63 913-922


















95-113





3420



361-368




139



675107-5112




Microbiol 69 275-84









Germany p129-180






















140

















3127-3133





7451ndash458






28 567












141







90









New York NY













15





157 174-209



42 252-258


142









Proteobacteria

Capiacutetulo







4


145

Abstract

















147

Introduction




































148


























Experimental design







149










microorganisms)










C2PL05

T5 Biostimulation

+ Bioaugmentation
















150

































151





































152

















136 and 145 days

Equation 2




Results











153

October

November

DecemberJanuary

FebruaryMarch

April MayJune

468

10121416182022

0 day

40 day

145 day

176 day

6 dayT

empe

ratu

re (

ordmC)

Month










6 40 145 17600

10x10-4

20x10-4

30x10-4

40x10-4

50x10-4

a a

b

aCO

2 mol

esg

of

soil

Time (days)



(plt005 SNK)


154

Toxicity assays











0 6 20 40 56 77 84 91 98 1051121251321411760

10

20

30

40

50

60

70

80

90

100 BA

Tox

icity

(

)


c

c

c

b

c

bc

bcbc

aa

aa

Treatment







155













156

T1 T2 T3 T4 T50000

0005

0010

0015

0020

0025

0030

0035

0040

g cr

eoso

te

g so

il


102030405060708090

100

C

aab

abb

a

bb

B

A

Ave

rage

res

idua

l con

cenr

atio

n of

PA

H (

)

T2 T3 T4 T50

102030405060708090

100

Tot

al r

esid

ual c

once

ntra

tion

of

PA

H (

)








157





Factor df SS F P




Error 20 14-5




Error 12 49143


PAH 2 168113 1452


Error 12 69486

p-value lt 005

p-value lt 001

p-value lt 0001


degradation















158













145 days


3 DUB-12RS

DUB-17RS 2875

16 DUB-17RS 1826

17 DUB-12RS

DUB-16RS 1414




176 days




26 DUB-13RS 1296




159






bands cloning


160



and 176 days (B)

















161

















162








163

Discussion















Colwell 1990)













contaminant







164






































165







166

References


































3411-3420


167







































168








3127-3133


























15





169




Orsis 13 105-117









42 252-258





Int 32 149-154





II


173



biodegradacioacuten
































174







































175



































176























1b)














177


































degradadora


178




































179






































180































III


183






























184














IV


187




























2078-2084




Microbiol 30 31-71


351-368


188









4895-113









1401-1405








580



3420









189






5112




Microbiol 69 275-84




10 208-217






















Germany p129-180




190





France























Technol 33 1552-1558










191






3542










3127-3133








Murcia



1942



Microbiol 152 45-49








192






























48-62








193






















Biochem 38 1125-1132









Biochem 40 3296-3302









194



174-178



Pollut 139 1-13





Technol 30136-142












Agradecimientos

197

Agradecimientos






























198


































199
























Raquel

Resumen



I


13

Antecedentes






















contaminados








14

























15


1996)


























1500000









16











ambiental posible

















Temperatura y pH




17









Kaushik 2009)






























18




































19





































20





































21





































22




































23








Resumen Objetivos

25

Objetivos






























27
















JA









28


M






29





































30






bacteriano C2PL05
































31










contaminante

























33




















muerta




A B


34

















naturales











35


Optimizacioacuten

CNP

BHB tween-80


antraceno

X 3

100101

1002116

100505

Optimizacioacuten

fuente de N

BHB tween-80


antraceno

X 3

NaNO3

NH4NO3

(NH4)2SO3

Optimizacioacuten

fuente de Fe

BHB tween-80


antraceno

X 3

FeCl3

Fe(NO3)3

Fe2(SO4)3

Optimizacioacuten

[Fe]

BHB tween-80


antraceno

X 3

005 mM

01 mM

02 mM

Optimizacioacuten

pH

BHB tween-80


antraceno

X 3

50

70

80

Optimizacioacuten

fuente de C

BHB tween-80

C2PL05



X 3

HAP

HAPglucosa (5050)

Glucosa

2ordm 3ordm

4ordm 5ordm 6ordm



36

Tordf









1001011002116100505

NaNO3

NH4NO3

(NH4)2SO3

FeCl3Fe(NO3)3

Fe2(SO4)3

005 mM01 mM02 mM

CMC20 CMC

10-1

10-2

10-3

0100505020100

18 tratamientos

X 3












X 3

X 3



el capiacutetulo 2


37














38

Tratamiento 1

Tratamiento 2

Tratamiento 3

Tratamiento 4









X 3

X 3

X 3

X 3

X 5 tiempos

X 5 tiempos

X 5 tiempos

X 5 tiempos
















39







natural




y Bioaumento






Suelo natural +X 2

Suelo natural +X 2

Suelo natural +X 2

Suelo natural +X 2

Suelo natural +X 2




40





contaminado




pH - 77 plusmn 01


WHCa v 33 plusmn 7


















consorcio










41




























42













(ordmC)











43






Tordfhibridc


Tordf finalf

4 ordmC infin

95 ordmC 5 min

95 ordmC 1 min

54 ordmC 05 min

72 ordmC 15 min

72 ordmC 10 min

30 CICLOS

PROGRAMA PCR III


Tordfhibridc


Tordf finalf

4 ordmC infin

95 ordmC 9 min

94 ordmC 1 min

55 ordmC 1 min

72 ordmC 15 min

72 ordmC 5 min

30 CICLOS

PROGRAMA PCR II


Tordfhibridc


Tordf finalf

4 ordmC infin

94 ordmC 9 min

94 ordmC 1 min

55 ordmC 1 min

72 ordmC 15 min

72 ordmC 5 min

30 CICLOS

PROGRAMA PCR I


44


Escherichia coli








comunidad
























45



































Capiacutetulo







1a


49

Abstract





















and so the costs


51

Introduction



































52



degradation rate


Chemicals and media




























53














Experimental design


















growth


54




















Bacterial growth





equation

1

1

ii

iii tt





55

Kinetic degradation







iBiiAii

i CkCkdt

dCr Eq 2




each PAH








was inoculated









56

Results







0 100 200 300 400 500 600 700

20

40

60

80

100

Rem

aini

ng P

AH

(

)

Time (hour)





CDI kB









pH 2 30middot10-2 1103 4 15middot10-4 5






57













171819202122232425

100101 1002116100505

bb

a

A

CNP molar ratio

CD

I


-30

-25

-20

-15

-10

-05

00B

d

g

e

bc

f

ab

f

Log

k B (

h-1)





deviation


58








19

20

21

22

23

24

25

(NH4)

2SO

4NH4NO

3NaNO

3

a

b

a

A

Nitrogen source

CD

I


2x10-3

4x10-3

6x10-3

8x10-3

1x10-2

Bf

ba

e

bcb

dbc

a

kB (

h-1)






59











168

172

176

180

184

188

192

196

Fe(NO3)

3 Fe2(SO

4)

3FeCl

3

ab

b

a

A

Iron source

CD

I


2x10-3

4x10-3

6x10-3

8x10-3

1x10-2

B

c

a

b

c

b

d

b

a a

k B

(h-1

)






60










005 01 02

38

40

42

44

46

48

50

a

a

a

A


CD

I


50x10-3

10x10-2

15x10-2

20x10-2

B

c

f

d

b

e

d

cb

a

k B (

h-1)





standard deviation


61









5 7 8

215

220

225

230

235

240

245

a

b

a

A

pH

CD

I


50x10-3

10x10-2

15x10-2

20x10-2

25x10-2

30x10-2

B

b

a

ab ab

a

ab

c

ab ab

kB

(h-1

)






62










source

PAHs (100)


18

20

22

24

26

28

Carbon source

b

c

a

A

CD

I


2x10-2

4x10-2

6x10-2

8x10-2

1x10-1

B

c

bb

b

b

a

k B (h

-1)







63

Discussion


































64



























associated costs

Acknowledgements






65

References




Biodegr 63 913-922








13











269-281





1612-1614







66







Elsevier








Dordrecht pp 1-23




5548










174-178



Soil Poll 13 1-13



Capiacutetulo





experimental design



1b


69

Abstract









71

Introduction



































72


degradation
















Chemicals and media










73


Treatment T

(ordmC) CNP (molar)

N source

Fe

source


(mM)

Glucose PAH ()


Inoculum dilution

1 30 100505 (NH4)2SO3 Fe2(SO4)3 02 0100 CMC 10-3

2 20 1002116 (NH4)2SO3 FeNO3 005 0100 + 20CMC 10-2

3 25 100101 NaNO3 FeNO3 02 0100 + 20CMC 10-1

4 20 100505 NaNO3 Fe2(SO4)3 02 5050 + 20CMC 10-2

5 25 100505 NH4NO3 FeNO3 01 5050 CMC 10-2

6 30 100101 NH4NO3 Fe2(SO4)3 005 8020 + 20CMC 10-2

7 30 100101 NaNO3 FeCl3 01 0100 CMC 10-2

8 20 100505 NaNO3 FeCl3 005 8020 CMC 10-1

9 25 1002116 (NH4)2SO3 FeCl3 02 8020 CMC 10-2

10 20 1002116 NH4NO3 Fe2(SO4)3 01 0100 CMC 10-1

11 20 100101 NH4NO3 FeNO3 02 8020 CMC 10-3

12 25 100101 (NH4)2SO3 Fe2(SO4)3 005 5050 CMC 10-1

13 25 1002116 NaNO3 Fe2(SO4)3 01 8020 + 20CMC 10-3

14 30 1002116 NH4NO3 FeCl3 02 5050 + 20CMC 10-1

15 25 100505 NH4NO3 FeCl3 005 0100 + 20CMC 10-3

16 30 1002116 NaNO3 FeNO3 005 5050 CMC 10-3

17 30 100505 (NH4)2SO3 FeNO3 01 8020 + 20CMC 10-1

18 20 100101 (NH4)2SO3 FeCl3 01 5050 + 20CMC 10-3











Experimental design





74












Cell density





1

1

ii

iii tt















75






















76




each treatment


1 965 883 864 904 2 969 950 833 917 3 966 895 845 902 4 972 915 921 872 5 969 904 950 882 6 982 935 995 852 7 964 883 859 902 8 977 953 964 823 9 976 936 984 825 10 970 910 895 925 11 979 968 986 888 12 966 889 920 850 13 978 930 993 835 14 966 897 943 871 15 963 881 898 914 16 963 886 951 867 17 977 954 986 861 18 976 930 967 915



100

50

5

100

101

100

211

6

CNP

20

ordmC

25ordmC

30ordmC

82

84

86

88

90

92 T (ordmC)

aa

a

aa

aa

aa

a

Tot

al P

D (

)

NaN

O3

NH

4NO

3

(NH

4)2S

O3

N source

FeC

L3

FeN

O3

Fe2

(SO

4)3

a

a

0acute05 0acute1

0acute2

Fe source

a

a

a

0 -

100

50 -

50

80 -

20

C Fe (mM)

a

b

c

CM

C

+ 2

0 C

MC

Gluc-PAHs

aa

10^-

1

10^-

2

10^-

3DilutionCMC

aa

a





77





T (ordmC) Error

2 056 1889 2 22 183 ns

51 002 51 12


2 003 069 ns 2 22 183 ns

51 005 51 12

N source Error

2 001 007 ns 2 214 177 ns 51 005 51 121

Fe source Error

2 003 066 ns 2 89 071 ns

51 005 51 126


2 007 146 ns 2 118 095 ns 51 005 51 124

Glucose-PAH Error

2 024 584 2 1802

3085 51 004 51 395

8

CMC Error

1 001 027 ns 1 89 071 ns

52 005 52 125


2 331 a 2 113 091 ns 54 6614 51 125


median = 044

p-value lt 001

p-value lt 0001

100

50

5

100

100

1

100

211

6

CNP

20

ordmC

25ordmC

30ordmC

16

17

18

19

20

21

a

a

aa

a

aa

a

c

bCD

I

NaN

O3

NH

4NO

3

(NH

4)2S

O3

N source

FeC

L3

FeN

O3

Fe2

SO

4

Fe source

a

a

0acute05 0acute1

0acute2

C Fe (mM)

a

a

a

0-10

0

50-5

0

80-2

0

Gluc-PAH

a

b

c

CM

C

+ 2

0 C

MC

CMC

aa

10^-

1

10^-

2

10^-

3

00

05

10

15

20

25

30

35C

DI n

orm

aliz

ed

DilutionT (ordmC)

b

a

a





78
























solubility














79



































can grow vigorously

80









only PAH

Acknowledgements






81

References















5112






1612-1614







1380







8 315-323

82












Dordrecht pp 1-23

























Poll 139 1-13


83



4 252-258



Capiacutetulo







2


87

Abstract















89

Introduction




































90



























Chemicals and media








91

































92







is avoided

SK

S

S

max

(Equation 1)




Xdt

dX (Equation 2)

















93










PAH monitoring








iBiiAii

i CkCkdt

dCr (Equation 3)














94


































95




process



















code DUB











96






























97

00

02

04

06

08

10

12

14

16

18

0 5 10 15 20 25 30 35 40 45

30

40

50

60

70

80

90

100

Tox

icity

(

)

Time (day)

B

A

Abs

orba

nce 60

0 nm

(A

U)







98








0 5 10 15 20 25 30 35 40 45

0

10

20

30

40

50

60

70

80

90

100

Res

idua

l con

cent

ratio

n of

PA

Hs

()

Time (days)

















99







Error 0021 2


PAH 15middot10-4 2 779 0001





























100
























101










102















DUB Band














103


















104

0 15 30

0102030405060708090

100

102030405060708090

100

D

C

B

A

0 15 30

F DIC-1JA DIC-2JA

E

G DIC-6JA DIC-5JA

0 15 30

H

Time (day)


Pse

udom

onas

ribot

ypes

(

)


102030405060708090

100

Ste

notr

opho

mon

as

ribot

ypes

(

)

DIC-6JA

0 15 30

102030405060708090

100

Ent

erob

acte

r rib

otyp

es (

)

DIC-4RS

Time (days)

Tot

al s

trai

ns (

)








105


degradation




































106




















107












108




30 2469 19

24 881 3447

27 845

21 516










Conclusions











109

Acknowledgment








110

References



































111



212



213ndash221




839-844






























112







647-6554














Capiacutetulo







3


115

Abstract
















117

Introcuduction



































118
















of consortium used


Chemicals and media














119
































120

1

1

ii

iii tt
































121


































122


used as out-group


degrading process





























123









Results






obtained

0 5 10 15 20 25 30 35

BO S08

C 2PL05

tim e (m in)




124






















125

0 11 33 66 101 137

005

010

015

020

025

030

035

0 11 33 66 101 137

0 33 101 137102

103

104

105

106

107

108

109


Time (day)

Abs

orba

nce 6

00nm

(A

U)

Time (day)

DC

BA

cell

g so

il




temperature range


126





μ


C2PL05 H 158 b 09 C2PL05 L 105 a 17

BOS08 H 241 c 17

BOS08 L 189 b 12









Factor df SS F

p-value

A) μ



Error 8 49 x 10-5 0001



Error 8 1281



PAH d 1 85098 251


Error 16 54225





127





0 11 33 66 101 137

0

20

40

60

80

100

0 11 33 66 101 137

BA

Time (day)

Tox

icity

(

)

Time (day)

















128












129



















130




(genera) 33

C2PL05

15 ordmC-5 ordmC



25 ordmC-15 ordmC



BOS08

15 ordmC-5 ordmC



25 ordmC-15 ordmC



101

C2PL05

15ordmC-5ordmC



25 ordmC-15 ordmC



BOS08

15 ordmC-5 ordmC




131

25 ordmC-15 ordmC




biodegradation




























132







BOS08 consortium



by SIMPER method



22 DUB-25RS 2855 2789 2581 20 DUB-24RS 2993 2521 1797 2366


4 Unidentified 2855 1120 2362 1755 2315 175

27 Unidentified 139

2 Unidentified 1198

24 DUB-26RS 929




133






101


134





Discussion


















135











toxicity were high



























136




































137










Acknowledgements




138

References





Biodegr 63 913-922


















95-113





3420



361-368




139



675107-5112




Microbiol 69 275-84









Germany p129-180






















140

















3127-3133





7451ndash458






28 567












141







90









New York NY













15





157 174-209



42 252-258


142









Proteobacteria

Capiacutetulo







4


145

Abstract

















147

Introduction




































148


























Experimental design







149










microorganisms)










C2PL05

T5 Biostimulation

+ Bioaugmentation
















150

































151





































152

















136 and 145 days

Equation 2




Results











153

October

November

DecemberJanuary

FebruaryMarch

April MayJune

468

10121416182022

0 day

40 day

145 day

176 day

6 dayT

empe

ratu

re (

ordmC)

Month










6 40 145 17600

10x10-4

20x10-4

30x10-4

40x10-4

50x10-4

a a

b

aCO

2 mol

esg

of

soil

Time (days)



(plt005 SNK)


154

Toxicity assays











0 6 20 40 56 77 84 91 98 1051121251321411760

10

20

30

40

50

60

70

80

90

100 BA

Tox

icity

(

)


c

c

c

b

c

bc

bcbc

aa

aa

Treatment







155













156

T1 T2 T3 T4 T50000

0005

0010

0015

0020

0025

0030

0035

0040

g cr

eoso

te

g so

il


102030405060708090

100

C

aab

abb

a

bb

B

A

Ave

rage

res

idua

l con

cenr

atio

n of

PA

H (

)

T2 T3 T4 T50

102030405060708090

100

Tot

al r

esid

ual c

once

ntra

tion

of

PA

H (

)








157





Factor df SS F P




Error 20 14-5




Error 12 49143


PAH 2 168113 1452


Error 12 69486

p-value lt 005

p-value lt 001

p-value lt 0001


degradation















158













145 days


3 DUB-12RS

DUB-17RS 2875

16 DUB-17RS 1826

17 DUB-12RS

DUB-16RS 1414




176 days




26 DUB-13RS 1296




159






bands cloning


160



and 176 days (B)

















161

















162








163

Discussion















Colwell 1990)













contaminant







164






































165







166

References


































3411-3420


167







































168








3127-3133


























15





169




Orsis 13 105-117









42 252-258





Int 32 149-154





II


173



biodegradacioacuten
































174







































175



































176























1b)














177


































degradadora


178




































179






































180































III


183






























184














IV


187




























2078-2084




Microbiol 30 31-71


351-368


188









4895-113









1401-1405








580



3420









189






5112




Microbiol 69 275-84




10 208-217






















Germany p129-180




190





France























Technol 33 1552-1558










191






3542










3127-3133








Murcia



1942



Microbiol 152 45-49








192






























48-62








193






















Biochem 38 1125-1132









Biochem 40 3296-3302









194



174-178



Pollut 139 1-13





Technol 30136-142












Agradecimientos

197

Agradecimientos






























198


































199
























Raquel


13

Antecedentes






















contaminados








14

























15


1996)


























1500000









16











ambiental posible

















Temperatura y pH




17









Kaushik 2009)






























18




































19





































20





































21





































22




































23








Resumen Objetivos

25

Objetivos






























27
















JA









28


M






29





































30






bacteriano C2PL05
































31










contaminante

























33




















muerta




A B


34

















naturales











35


Optimizacioacuten

CNP

BHB tween-80


antraceno

X 3

100101

1002116

100505

Optimizacioacuten

fuente de N

BHB tween-80


antraceno

X 3

NaNO3

NH4NO3

(NH4)2SO3

Optimizacioacuten

fuente de Fe

BHB tween-80


antraceno

X 3

FeCl3

Fe(NO3)3

Fe2(SO4)3

Optimizacioacuten

[Fe]

BHB tween-80


antraceno

X 3

005 mM

01 mM

02 mM

Optimizacioacuten

pH

BHB tween-80


antraceno

X 3

50

70

80

Optimizacioacuten

fuente de C

BHB tween-80

C2PL05



X 3

HAP

HAPglucosa (5050)

Glucosa

2ordm 3ordm

4ordm 5ordm 6ordm



36

Tordf









1001011002116100505

NaNO3

NH4NO3

(NH4)2SO3

FeCl3Fe(NO3)3

Fe2(SO4)3

005 mM01 mM02 mM

CMC20 CMC

10-1

10-2

10-3

0100505020100

18 tratamientos

X 3












X 3

X 3



el capiacutetulo 2


37














38

Tratamiento 1

Tratamiento 2

Tratamiento 3

Tratamiento 4









X 3

X 3

X 3

X 3

X 5 tiempos

X 5 tiempos

X 5 tiempos

X 5 tiempos
















39







natural




y Bioaumento






Suelo natural +X 2

Suelo natural +X 2

Suelo natural +X 2

Suelo natural +X 2

Suelo natural +X 2




40





contaminado




pH - 77 plusmn 01


WHCa v 33 plusmn 7


















consorcio










41




























42













(ordmC)











43






Tordfhibridc


Tordf finalf

4 ordmC infin

95 ordmC 5 min

95 ordmC 1 min

54 ordmC 05 min

72 ordmC 15 min

72 ordmC 10 min

30 CICLOS

PROGRAMA PCR III


Tordfhibridc


Tordf finalf

4 ordmC infin

95 ordmC 9 min

94 ordmC 1 min

55 ordmC 1 min

72 ordmC 15 min

72 ordmC 5 min

30 CICLOS

PROGRAMA PCR II


Tordfhibridc


Tordf finalf

4 ordmC infin

94 ordmC 9 min

94 ordmC 1 min

55 ordmC 1 min

72 ordmC 15 min

72 ordmC 5 min

30 CICLOS

PROGRAMA PCR I


44


Escherichia coli








comunidad
























45



































Capiacutetulo







1a


49

Abstract





















and so the costs


51

Introduction



































52



degradation rate


Chemicals and media




























53














Experimental design


















growth


54




















Bacterial growth





equation

1

1

ii

iii tt





55

Kinetic degradation







iBiiAii

i CkCkdt

dCr Eq 2




each PAH








was inoculated









56

Results







0 100 200 300 400 500 600 700

20

40

60

80

100

Rem

aini

ng P

AH

(

)

Time (hour)





CDI kB









pH 2 30middot10-2 1103 4 15middot10-4 5






57













171819202122232425

100101 1002116100505

bb

a

A

CNP molar ratio

CD

I


-30

-25

-20

-15

-10

-05

00B

d

g

e

bc

f

ab

f

Log

k B (

h-1)





deviation


58








19

20

21

22

23

24

25

(NH4)

2SO

4NH4NO

3NaNO

3

a

b

a

A

Nitrogen source

CD

I


2x10-3

4x10-3

6x10-3

8x10-3

1x10-2

Bf

ba

e

bcb

dbc

a

kB (

h-1)






59











168

172

176

180

184

188

192

196

Fe(NO3)

3 Fe2(SO

4)

3FeCl

3

ab

b

a

A

Iron source

CD

I


2x10-3

4x10-3

6x10-3

8x10-3

1x10-2

B

c

a

b

c

b

d

b

a a

k B

(h-1

)






60










005 01 02

38

40

42

44

46

48

50

a

a

a

A


CD

I


50x10-3

10x10-2

15x10-2

20x10-2

B

c

f

d

b

e

d

cb

a

k B (

h-1)





standard deviation


61









5 7 8

215

220

225

230

235

240

245

a

b

a

A

pH

CD

I


50x10-3

10x10-2

15x10-2

20x10-2

25x10-2

30x10-2

B

b

a

ab ab

a

ab

c

ab ab

kB

(h-1

)






62










source

PAHs (100)


18

20

22

24

26

28

Carbon source

b

c

a

A

CD

I


2x10-2

4x10-2

6x10-2

8x10-2

1x10-1

B

c

bb

b

b

a

k B (h

-1)







63

Discussion


































64



























associated costs

Acknowledgements






65

References




Biodegr 63 913-922








13











269-281





1612-1614







66







Elsevier








Dordrecht pp 1-23




5548










174-178



Soil Poll 13 1-13



Capiacutetulo





experimental design



1b


69

Abstract









71

Introduction



































72


degradation
















Chemicals and media










73


Treatment T

(ordmC) CNP (molar)

N source

Fe

source


(mM)

Glucose PAH ()


Inoculum dilution

1 30 100505 (NH4)2SO3 Fe2(SO4)3 02 0100 CMC 10-3

2 20 1002116 (NH4)2SO3 FeNO3 005 0100 + 20CMC 10-2

3 25 100101 NaNO3 FeNO3 02 0100 + 20CMC 10-1

4 20 100505 NaNO3 Fe2(SO4)3 02 5050 + 20CMC 10-2

5 25 100505 NH4NO3 FeNO3 01 5050 CMC 10-2

6 30 100101 NH4NO3 Fe2(SO4)3 005 8020 + 20CMC 10-2

7 30 100101 NaNO3 FeCl3 01 0100 CMC 10-2

8 20 100505 NaNO3 FeCl3 005 8020 CMC 10-1

9 25 1002116 (NH4)2SO3 FeCl3 02 8020 CMC 10-2

10 20 1002116 NH4NO3 Fe2(SO4)3 01 0100 CMC 10-1

11 20 100101 NH4NO3 FeNO3 02 8020 CMC 10-3

12 25 100101 (NH4)2SO3 Fe2(SO4)3 005 5050 CMC 10-1

13 25 1002116 NaNO3 Fe2(SO4)3 01 8020 + 20CMC 10-3

14 30 1002116 NH4NO3 FeCl3 02 5050 + 20CMC 10-1

15 25 100505 NH4NO3 FeCl3 005 0100 + 20CMC 10-3

16 30 1002116 NaNO3 FeNO3 005 5050 CMC 10-3

17 30 100505 (NH4)2SO3 FeNO3 01 8020 + 20CMC 10-1

18 20 100101 (NH4)2SO3 FeCl3 01 5050 + 20CMC 10-3











Experimental design





74












Cell density





1

1

ii

iii tt















75






















76




each treatment


1 965 883 864 904 2 969 950 833 917 3 966 895 845 902 4 972 915 921 872 5 969 904 950 882 6 982 935 995 852 7 964 883 859 902 8 977 953 964 823 9 976 936 984 825 10 970 910 895 925 11 979 968 986 888 12 966 889 920 850 13 978 930 993 835 14 966 897 943 871 15 963 881 898 914 16 963 886 951 867 17 977 954 986 861 18 976 930 967 915



100

50

5

100

101

100

211

6

CNP

20

ordmC

25ordmC

30ordmC

82

84

86

88

90

92 T (ordmC)

aa

a

aa

aa

aa

a

Tot

al P

D (

)

NaN

O3

NH

4NO

3

(NH

4)2S

O3

N source

FeC

L3

FeN

O3

Fe2

(SO

4)3

a

a

0acute05 0acute1

0acute2

Fe source

a

a

a

0 -

100

50 -

50

80 -

20

C Fe (mM)

a

b

c

CM

C

+ 2

0 C

MC

Gluc-PAHs

aa

10^-

1

10^-

2

10^-

3DilutionCMC

aa

a





77





T (ordmC) Error

2 056 1889 2 22 183 ns

51 002 51 12


2 003 069 ns 2 22 183 ns

51 005 51 12

N source Error

2 001 007 ns 2 214 177 ns 51 005 51 121

Fe source Error

2 003 066 ns 2 89 071 ns

51 005 51 126


2 007 146 ns 2 118 095 ns 51 005 51 124

Glucose-PAH Error

2 024 584 2 1802

3085 51 004 51 395

8

CMC Error

1 001 027 ns 1 89 071 ns

52 005 52 125


2 331 a 2 113 091 ns 54 6614 51 125


median = 044

p-value lt 001

p-value lt 0001

100

50

5

100

100

1

100

211

6

CNP

20

ordmC

25ordmC

30ordmC

16

17

18

19

20

21

a

a

aa

a

aa

a

c

bCD

I

NaN

O3

NH

4NO

3

(NH

4)2S

O3

N source

FeC

L3

FeN

O3

Fe2

SO

4

Fe source

a

a

0acute05 0acute1

0acute2

C Fe (mM)

a

a

a

0-10

0

50-5

0

80-2

0

Gluc-PAH

a

b

c

CM

C

+ 2

0 C

MC

CMC

aa

10^-

1

10^-

2

10^-

3

00

05

10

15

20

25

30

35C

DI n

orm

aliz

ed

DilutionT (ordmC)

b

a

a





78
























solubility














79



































can grow vigorously

80









only PAH

Acknowledgements






81

References















5112






1612-1614







1380







8 315-323

82












Dordrecht pp 1-23

























Poll 139 1-13


83



4 252-258



Capiacutetulo







2


87

Abstract















89

Introduction




































90



























Chemicals and media








91

































92







is avoided

SK

S

S

max

(Equation 1)




Xdt

dX (Equation 2)

















93










PAH monitoring








iBiiAii

i CkCkdt

dCr (Equation 3)














94


































95




process



















code DUB











96






























97

00

02

04

06

08

10

12

14

16

18

0 5 10 15 20 25 30 35 40 45

30

40

50

60

70

80

90

100

Tox

icity

(

)

Time (day)

B

A

Abs

orba

nce 60

0 nm

(A

U)







98








0 5 10 15 20 25 30 35 40 45

0

10

20

30

40

50

60

70

80

90

100

Res

idua

l con

cent

ratio

n of

PA

Hs

()

Time (days)

















99







Error 0021 2


PAH 15middot10-4 2 779 0001





























100
























101










102















DUB Band














103


















104

0 15 30

0102030405060708090

100

102030405060708090

100

D

C

B

A

0 15 30

F DIC-1JA DIC-2JA

E

G DIC-6JA DIC-5JA

0 15 30

H

Time (day)


Pse

udom

onas

ribot

ypes

(

)


102030405060708090

100

Ste

notr

opho

mon

as

ribot

ypes

(

)

DIC-6JA

0 15 30

102030405060708090

100

Ent

erob

acte

r rib

otyp

es (

)

DIC-4RS

Time (days)

Tot

al s

trai

ns (

)








105


degradation




































106




















107












108




30 2469 19

24 881 3447

27 845

21 516










Conclusions











109

Acknowledgment








110

References



































111



212



213ndash221




839-844






























112







647-6554














Capiacutetulo







3


115

Abstract
















117

Introcuduction



































118
















of consortium used


Chemicals and media














119
































120

1

1

ii

iii tt
































121


































122


used as out-group


degrading process





























123









Results






obtained

0 5 10 15 20 25 30 35

BO S08

C 2PL05

tim e (m in)




124






















125

0 11 33 66 101 137

005

010

015

020

025

030

035

0 11 33 66 101 137

0 33 101 137102

103

104

105

106

107

108

109


Time (day)

Abs

orba

nce 6

00nm

(A

U)

Time (day)

DC

BA

cell

g so

il




temperature range


126





μ


C2PL05 H 158 b 09 C2PL05 L 105 a 17

BOS08 H 241 c 17

BOS08 L 189 b 12









Factor df SS F

p-value

A) μ



Error 8 49 x 10-5 0001



Error 8 1281



PAH d 1 85098 251


Error 16 54225





127





0 11 33 66 101 137

0

20

40

60

80

100

0 11 33 66 101 137

BA

Time (day)

Tox

icity

(

)

Time (day)

















128












129



















130




(genera) 33

C2PL05

15 ordmC-5 ordmC



25 ordmC-15 ordmC



BOS08

15 ordmC-5 ordmC



25 ordmC-15 ordmC



101

C2PL05

15ordmC-5ordmC



25 ordmC-15 ordmC



BOS08

15 ordmC-5 ordmC




131

25 ordmC-15 ordmC




biodegradation




























132







BOS08 consortium



by SIMPER method



22 DUB-25RS 2855 2789 2581 20 DUB-24RS 2993 2521 1797 2366


4 Unidentified 2855 1120 2362 1755 2315 175

27 Unidentified 139

2 Unidentified 1198

24 DUB-26RS 929




133






101


134





Discussion


















135











toxicity were high



























136




































137










Acknowledgements




138

References





Biodegr 63 913-922


















95-113





3420



361-368




139



675107-5112




Microbiol 69 275-84









Germany p129-180






















140

















3127-3133





7451ndash458






28 567












141







90









New York NY













15





157 174-209



42 252-258


142









Proteobacteria

Capiacutetulo







4


145

Abstract

















147

Introduction




































148


























Experimental design







149










microorganisms)










C2PL05

T5 Biostimulation

+ Bioaugmentation
















150

































151





































152

















136 and 145 days

Equation 2




Results











153

October

November

DecemberJanuary

FebruaryMarch

April MayJune

468

10121416182022

0 day

40 day

145 day

176 day

6 dayT

empe

ratu

re (

ordmC)

Month










6 40 145 17600

10x10-4

20x10-4

30x10-4

40x10-4

50x10-4

a a

b

aCO

2 mol

esg

of

soil

Time (days)



(plt005 SNK)


154

Toxicity assays











0 6 20 40 56 77 84 91 98 1051121251321411760

10

20

30

40

50

60

70

80

90

100 BA

Tox

icity

(

)


c

c

c

b

c

bc

bcbc

aa

aa

Treatment







155













156

T1 T2 T3 T4 T50000

0005

0010

0015

0020

0025

0030

0035

0040

g cr

eoso

te

g so

il


102030405060708090

100

C

aab

abb

a

bb

B

A

Ave

rage

res

idua

l con

cenr

atio

n of

PA

H (

)

T2 T3 T4 T50

102030405060708090

100

Tot

al r

esid

ual c

once

ntra

tion

of

PA

H (

)








157





Factor df SS F P




Error 20 14-5




Error 12 49143


PAH 2 168113 1452


Error 12 69486

p-value lt 005

p-value lt 001

p-value lt 0001


degradation















158













145 days


3 DUB-12RS

DUB-17RS 2875

16 DUB-17RS 1826

17 DUB-12RS

DUB-16RS 1414




176 days




26 DUB-13RS 1296




159






bands cloning


160



and 176 days (B)

















161

















162








163

Discussion















Colwell 1990)













contaminant







164






































165







166

References


































3411-3420


167







































168








3127-3133


























15





169




Orsis 13 105-117









42 252-258





Int 32 149-154





II


173



biodegradacioacuten
































174







































175



































176























1b)














177


































degradadora


178




































179






































180































III


183






























184














IV


187




























2078-2084




Microbiol 30 31-71


351-368


188









4895-113









1401-1405








580



3420









189






5112




Microbiol 69 275-84




10 208-217






















Germany p129-180




190





France























Technol 33 1552-1558










191






3542










3127-3133








Murcia



1942



Microbiol 152 45-49








192






























48-62








193






















Biochem 38 1125-1132









Biochem 40 3296-3302









194



174-178



Pollut 139 1-13





Technol 30136-142












Agradecimientos

197

Agradecimientos






























198


































199
























Raquel

Biorremediación de suelos - URJC

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